3-hydroxypropionic acid and other organic compounds

ABSTRACT

Methods and materials related to producing 3-HP as well as other organic compounds are disclosed. Specifically, isolated nucleic acids, polypeptides, host cells, and methods and materials for producing 3-HP and other organic compounds are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. application Ser. No. 11/539,856 filed Oct.9, 2006, which is a divisional of U.S. application Ser. No. 10/432,443filed Oct. 20, 2003 now U.S. Pat. No. 7,186,541, which is the NationalStage of International Application No. PCT/US01/43607, filed Nov. 20,2001, which in turn claims the benefit of U.S. Provisional ApplicationNo. 60/252,123 filed Nov. 20, 2000, 60/285,478 filed Apr. 20, 2001,60/306,727 filed Jul. 20, 2001, and 60/317,845 filed Sep. 7, 2001, allherein incorporated by reference.

FIELD OF THE INVENTION

The invention relates to enzymes and methods that can be used to produceorganic acids and related products.

BACKGROUND

Organic chemicals such as organic acids, esters, and polyols can be usedto synthesize plastic materials and other products. To meet theincreasing demand for organic chemicals, more efficient and costeffective production methods are being developed which utilize rawmaterials based on carbohydrates rather than hydrocarbons. For example,certain bacteria have been used to produce large quantities of lacticacid used in the production of polylactic acid.

3-hydroxypropionic acid (3-HP) is an organic acid. Although severalchemical synthesis routes have been described to produce 3-HP, only onebiocatalytic route has been heretofore previously disclosed (WO 01/16346to Suthers, et al.). 3-HP has utility for specialty synthesis and can beconverted to commercially important intermediates by known art in thechemical industry, e.g., acrylic acid by dehydration, malonic acid byoxidation, esters by esterification reactions with alcohols, andreduction to 1,3 propanediol.

SUMMARY

The invention relates to methods and materials involved in producing3-hydroxypropionic acid and other organic compounds (e.g.,1,3-propanediol, acrylic acid, polymerized acrylate, esters of acrylate,polymerized 3-HP, esters of 3-HP, and malonic acid and its esters).Specifically, the invention provides nucleic acid molecules,polypeptides, host cells, and methods that can be used to produce 3-HPand other organic compounds such as 1,3-propanediol, acrylic acid,polymerized acrylate, esters of acrylate, polymerized 3-HP, esters of3-HP, and malonic acid and its esters. 3-HP has potential to be bothbiologically and commercially important. For example, the nutritionalindustry can use 3-HP as a food, feed additive or preservative, whilethe derivatives mentioned above can be produced from 3-HP. The nucleicacid molecules described herein can be used to engineer host cells withthe ability to produce 3-HP as well as other organic compounds such as1,3-propanediol, acrylic acid, polymerized acrylate, esters of acrylate,polymerized 3-HP, and esters of 3-HP. The polypeptides described hereincan be used in cell-free systems to make 3-HP as well as other organiccompounds such as 1,3-propanediol, acrylic acid, polymerized acrylate,esters of acrylate, polymerized 3-HP, and esters of 3-HP. The host cellsdescribed herein can be used in culture systems to produce largequantities of 3-HP as well as other organic compounds such as1,3-propanediol, acrylic acid, polymerized acrylate, esters of acrylate,polymerized 3-HP, and esters of 3-HP.

One aspect of the invention provides cells that have lactyl-CoAdehydratase activity and 3-hydroxypropionyl-CoA dehydratase activity,and methods of making products such as those described herein byculturing at least one of the cells that have lactyl-CoA dehydrataseactivity and 3-hydroxypropionyl-CoA dehydratase activity. In someembodiments, the cell can also contain an exogenous nucleic acidmolecule that encodes one or more of the following polypeptides: apolypeptide having E1 activator activity; an E2 α polypeptide that is asubunit of an enzyme having lactyl-CoA dehydratase activity; an E2 βpolypeptide that is a subunit of an enzyme having lactyl-CoA dehydrataseactivity; and a polypeptide having 3-hydroxypropionyl-CoA dehydrataseactivity. Additionally, the cell can have CoA transferase activity, CoAsynthetase activity, poly hydroxyacid synthase activity,3-hydroxypropionyl-CoA hydrolase activity, 3-hydroxyisobutryl-CoAhydrolase activity, and/or lipase activity. Moreover, the cell cancontain at least one exogenous nucleic acid molecule that expresses oneor more polypeptides that have CoA transferase activity,3-hydroxypropionyl-CoA hydrolase activity, 3-hydroxyisobutryl-CoAhydrolase activity, CoA synthetase activity, poly hydroxyacid synthaseactivity, and/or lipase activity.

In another embodiment of the invention, the cell that has lactyl-CoAdehydratase activity and 3-hydroxypropionyl-CoA dehydratase activityproduces a product, for example, 3-HP, polymerized 3-HP, and/or an esterof 3-HP, such as methyl hydroxypropionate, ethyl hydroxypropionate,propyl hydroxypropionate, and/or butyl hydroxypropionate. Accordingly,the invention also provides methods of producing one or more of theseproducts. These methods involve culturing the cell that has lactyl-CoAdehydratase activity and 3-hydroxypropionyl-CoA dehydratase activityunder conditions that allow the product to be produced. These cells alsocan have CoA synthetase activity and/or poly hydroxyacid synthaseactivity.

Another aspect of the invention provides cells that have CoA synthetaseactivity, lactyl-CoA dehydratase activity, and poly hydroxyacid synthaseactivity. In some embodiments, these cells also can contain an exogenousnucleic acid molecule that encodes one or more of the followingpolypeptides: a polypeptide having E1 activator activity; an E2 αpolypeptide that is a subunit of an enzyme having lactyl-CoA dehydrataseactivity; an E2 β polypeptide that is a subunit of an enzyme havinglactyl-CoA dehydratase activity; a polypeptide having CoA synthetaseactivity; and a polypeptide having poly hydroxyacid synthase activity.

In another embodiment of the invention, the cell that has CoA synthetaseactivity, lactyl-CoA dehydratase activity, and poly hydroxyacid synthaseactivity can produce a product, for example, polymerized acrylate.

Another aspect of the invention provides a cell comprising CoAtransferase activity, lactyl-CoA dehydratase activity, and lipaseactivity. In some embodiments, the cell also can contain an exogenousnucleic acid molecule that encodes one or more of the followingpolypeptides: a polypeptide having CoA transferase activity; apolypeptide having E1 activator activity; an E2 α polypeptide that is asubunit of an enzyme having lactyl-CoA dehydratase activity; an E2 βpolypeptide that is a subunit of an enzyme having lactyl-CoA dehydrataseactivity; and a polypeptide having lipase activity. This cell can beused, among other things, to produce products such as esters of acrylate(e.g., methyl acrylate, ethyl acrylate, propyl acrylate, and butylacrylate).

In some embodiments, 1,3 propanediol can be created from either 3-HP-CoAor 3-HP via the use of polypeptides having enzymatic activity. Thesepolypeptides can be used either in vitro or in vivo. When converting3-HP-CoA to 1,3 propanediol, polypeptides having oxidoreductase activityor reductase activity (e.g., enzymes from the 1.1.1.- class of enzymes)can be used. Alternatively, when creating 1,3 propanediol from 3-HP, acombination of (1) a polypeptide having aldehyde dehydrogenase activity(e.g., an enzyme from the 1.1.1.34 class) and (2) a polypeptide havingalcohol dehydrogenase activity (e.g., an enzyme from the 1.1.1.32 class)can be used.

In some embodiments of the invention, products are produced in vitro(outside of a cell). In other embodiments of the invention, products areproduced using a combination of in vitro and in vivo (within a cell)methods. In yet other embodiments of the invention, products areproduced in vivo. For methods involving in vivo steps, the cells can beisolated cultured cells or whole organisms such as transgenic plants,non-human mammals, or single-celled organisms such as yeast and bacteria(e.g., Lactobacillus, Lactococcus, Bacillus, and Escherichia cells).Hereinafter such cells are referred to as production cells. Productsproduced by these production cells can be organic products such as 3-HPand/or the nucleic acid molecules and polypeptides described herein.

Another aspect of the invention provides polypeptides having an aminoacid sequence that (1) is set forth in SEQ ID NO:2, 10, 18, 26, 35, 37,39, 41, 141, 160, or 161, (2) is at least 10 contiguous amino acidresidues of a sequence set forth in SEQ ID NO:2, 10, 18, 26, 35, 37, 39,41, 141, 160, or 161, (3) has at least 65 percent sequence identity withat least 10 contiguous amino acid residues of a sequence set forth inSEQ ID NO:2, 10, 18, 26, 35, 37, 39, 41, 141, 160, or 161, (4) is asequence set forth in SEQ ID NO:2, 10, 18, 26, 35, 37, 39, 41, 141, 160,or 161 having conservative amino acid substitutions, or (5) has at least65 percent sequence identity with a sequence set forth in SEQ ID NO:2,10, 18, 26, 35, 37, 39, 41, 141, 160, or 161. Accordingly, the inventionalso provides nucleic acid sequences that encode any of the polypeptidesdescribed herein as well as specific binding agents that bind to any ofthe polypeptides described herein. Likewise, the invention providestransformed cells that contain any of the nucleic acid sequences thatencode any of the polypeptides described herein. These cells can be usedto produce nucleic acid molecules, polypeptides, and organic compounds.The polypeptides can be used to catalyze the formation of organiccompounds or can be used as antigens to create specific binding agents.

In yet another embodiment, the invention provides isolated nucleic acidmolecules that contain at least one of the following nucleic acidsequences: (1) a nucleic acid sequence as set forth in SEQ ID NO:1, 9,17, 25, 33, 34, 36, 38, 40, 42, 129, 140, 142, 162, or 163; (2) anucleic acid sequence having at least 10 consecutive nucleotides from asequence set forth in SEQ ID NO:1, 9, 17, 25, 33, 34, 36, 38, 40, 42,129, 140, 142, 162, or 163; (3) a nucleic acid sequences that hybridizeunder hybridization conditions (e.g., moderately or highly stringenthybridization conditions) to a sequence set forth in SEQ ID NO:1, 9, 17,25, 33, 34, 36, 38, 40, 42, 129, 140, 142, 162, or 163; (4) a nucleicacid sequence having 65 percent sequence identity with at least 10consecutive nucleotides from a sequence set forth in SEQ ID NO:1, 9, 17,25, 33, 34, 36, 38, 40, 42, 129, 140, 142, 162, or 163; and (5) anucleic acid sequence having at least 65 percent sequence identity witha sequence set forth in SEQ ID NO:1, 9, 17, 25, 33, 34, 36, 38, 40, 42,129, 140, 142, 162, or 163. Accordingly, the invention also provides aproduction cell that contains at least one exogenous nucleic acid havingany the nucleic acid sequences provided above. The production cell canbe used to express polypeptides that have an enzymatic activity such asCoA transferase activity, lactyl-CoA dehydratase activity, CoA synthaseactivity, dehydratase activity, dehydrogenase activity, malonyl CoAreductase activity, β-alanine ammonia lyase activity, and/or3-hydroxypropionyl-CoA dehydratase activity. Accordingly, the inventionalso provides methods of producing polypeptides encoded by the nucleicacid sequences described above.

The invention also provides several methods such as methods for making3-HP from lactate, phosphoenolpyruvate (PEP), or pyruvate. In someembodiments, methods for making 3-HP from lactate, PEP, or pyruvateinvolve culturing a cell containing at least one exogenous nucleic acidunder conditions that allow the cell to produce 3-HP. These methods canbe practiced using the various types of production cells describedherein. In some embodiments, the production cells can have one or moreof the following activities: CoA transferase activity,3-hydroxypropionyl-CoA hydrolase activity, 3-hydroxyisobutryl-CoAhydrolase activity, dehydratase activity, and/or malonyl CoA reductaseactivity.

In other embodiments, the methods involve making 3-HP wherein lactate iscontacted with a first polypeptide having CoA transferase activity orCoA synthetase activity such that lactyl-CoA is formed, then contactinglactyl-CoA with a second polypeptide having lactyl-CoA dehydrataseactivity to form acrylyl-CoA, then contacting acrylyl-CoA with a thirdpolypeptide having 3-hydroxypropionyl-CoA dehydratase activity to form3-hydroxypropionic acid-CoA, and then contacting 3-hydroxypropionicacid-CoA with the first polypeptide to form 3-HP or with a fourthpolypeptide having 3-hydroxypropionyl-CoA hydrolase activity or3-hydroxyisobutryl-CoA hydrolase activity to form 3-HP.

Another aspect of the invention provides methods for making polymerized3-HP. These methods involve making 3-hydroxypropionic acid-CoA asdescribed above, and then contacting the 3-hydroxypropionic acid-CoAwith a polypeptide having poly hydroxyacid synthase activity to formpolymerized 3-HP.

In yet another embodiment of the invention, methods for making an esterof 3-HP are provided. These methods involve making 3-HP as describedabove, and then additionally contacting 3-HP with a fifth polypeptidehaving lipase activity to form an ester.

The invention also provides methods for making polymerized acrylate.These methods involve culturing a cell that has both CoA synthetaseactivity, lactyl-CoA dehydratase activity, and poly hydroxyacid synthaseactivity such that polymerized acrylate is made. Accordingly, theinvention also provides methods of making polymerized acrylate whereinlactate is contacted with a first polypeptide having CoA synthetaseactivity to form lactyl-CoA, then contacting lactyl-CoA with a secondpolypeptide having lactyl-CoA dehydratase activity to form acrylyl-CoA,and then contacting acrylyl-CoA with a third polypeptide having polyhydroxyacid synthase activity to form polymerized acrylate.

The invention also provides methods of making an ester of acrylate.These methods involve culturing a cell that has CoA transferaseactivity, lipase activity, and lactyl-CoA dehydratase activity underconditions that allow the cell to produce an ester.

In another embodiment, the invention provides methods for making anester of acrylate, wherein acrylyl-CoA is formed as described above, andthen acrylyl-CoA is contacted with a polypeptide having CoA transferaseactivity to form acrylate, and acrylate is contacted with a polypeptidehaving lipase activity to form the ester.

The invention also provides methods for making 3-HP. These methodsinvolve culturing a cell containing at least one exogenous nucleic acidthat encodes at least one polypeptide such that 3-HP is produced fromacetyl-CoA or malonyl-CoA.

Alternative embodiments provide methods of making 3-HP, whereinacetyl-CoA is contacted with a first polypeptide having acetyl-CoAcarboxylase activity to form malonyl-CoA, and malonyl-CoA is contactedwith a second polypeptide having malonyl-CoA reductase activity to form3-HP.

In other embodiments, malonyl-CoA can be contacted with a polypeptidehaving malonyl-CoA reductase activity so that 3-HP can be made.

In another embodiment, the invention provides a method for making 3-HPthat uses a β-alanine intermediate. This method can be performed bycontacting β-alanine CoA with a first polypeptide having β-alanyl-CoAammonia lyase activity (such as a polypeptide having the amino acidsequence set forth in SEQ ID NO: 160 or 161) to form acrylyl-CoA,contacting acrylyl-CoA with a second polypeptide having 3-HP-CoAdehydratase activity to form 3-HP-CoA, and contacting 3-HP-CoA with athird polypeptide having glutamate dehydrogenase activity to make 3-HP.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram of a pathway for making 3-HP.

FIG. 2 is a diagram of a pathway for making polymerized 3-HP.

FIG. 3 is a diagram of a pathway for making esters of 3-HP.

FIG. 4 is a diagram of a pathway for making polymerized acrylic acid.

FIG. 5 is a diagram of a pathway for making esters of acrylate.

FIG. 6 is a listing of a nucleic acid sequence that encodes apolypeptide having CoA transferase activity (SEQ ID NO:1).

FIG. 7 is a listing of an amino acid sequence of a polypeptide havingCoA transferase activity (SEQ ID NO:2).

FIG. 8 is an alignment of the nucleic acid sequences set forth in SEQ IDNOs:1, 3, 4, and 5.

FIG. 9 is an alignment of the amino acid sequences set forth in SEQ IDNOs:2, 6, 7, and 8.

FIG. 10 is a listing of a nucleic acid sequence that encodes apolypeptide having E1 activator activity (SEQ ID NO:9).

FIG. 11 is a listing of an amino acid sequence of a polypeptide havingE1 activator activity (SEQ ID NO:10).

FIG. 12 is an alignment of the nucleic acid sequences set forth in SEQID NOs:9, 11, 12, and 13.

FIG. 13 is an alignment of the amino acid sequences set forth in SEQ IDNOs:10, 14, 15, and 16.

FIG. 14 is a listing of a nucleic acid sequence that encodes an E2 αsubunit of an enzyme having lactyl-CoA dehydratase activity (SEQ IDNO:17).

FIG. 15 is a listing of an amino acid sequence of an E2 α subunit of anenzyme having lactyl-CoA dehydratase activity (SEQ ID NO:18).

FIG. 16 is an alignment of the nucleic acid sequences set forth in SEQID NOs:17, 19, 20, and 21.

FIG. 17 is an alignment of the amino acid sequences set forth in SEQ IDNOs:18, 22, 23, and 24.

FIG. 18 is a listing of a nucleic acid sequence that encodes an E2 βsubunit of an enzyme having lactyl-CoA dehydratase activity (SEQ IDNO:25). The “G” at position 443 can be an “A”; and the “A” at position571 can be a “G”.

FIG. 19 is a listing of an amino acid sequence of an E2 β subunit of anenzyme having lactyl-CoA dehydratase activity (SEQ ID NO:26).

FIG. 20 is an alignment of the nucleic acid sequences set forth in SEQID NOs:25, 27, 28, and 29.

FIG. 21 is an alignment of the amino acid sequences set forth in SEQ IDNOs:26, 30, 31, and 32.

FIG. 22 is a listing of a nucleic acid sequence of genomic DNA fromMegasphaera elsdenii (SEQ ID NO:33).

FIG. 23 is a listing of a nucleic acid sequence that encodes apolypeptide from Megasphaera elsdenii (SEQ ID NO:34).

FIG. 24 is a listing of an amino acid sequence of a polypeptide fromMegasphaera elsdenii (SEQ ID NO:35).

FIG. 25 is a listing of a nucleic acid sequence that encodes apolypeptide having enzymatic activity (SEQ ID NO:36).

FIG. 26 is a listing of an amino acid sequence of a polypeptide havingenzymatic activity (SEQ ID NO:37).

FIG. 27 is a listing of a nucleic acid sequence that contains non-codingas well as coding sequence of a polypeptide having CoA synthase,dehydratase, and dehydrogenase activity (SEQ ID NO:38). The start sitefor the coding sequence is at position 480, a ribosome binding site isat position 466-473, and the stop codon is at position 5946.

FIG. 28 is a listing of an amino acid sequence from a polypeptide havingCoA synthase, dehydratase, and dehydrogenase activity (SEQ ID NO:39).

FIG. 29 is a listing of a nucleic acid sequence that encodes apolypeptide having 3-hydroxypropionyl-CoA dehydratase activity (SEQ IDNO:40).

FIG. 30 is a listing of an amino acid sequence of a polypeptide having3-hydroxypropionyl-CoA dehydratase activity (SEQ ID NO:41).

FIG. 31 is a listing of a nucleic acid sequence that contains non-codingas well as coding sequence of a polypeptide having3-hydroxypropionyl-CoA dehydratase activity (SEQ ID NO:42).

FIG. 32 is an alignment of the nucleic acid sequences set forth in SEQID NOs:40, 43, 44, and 45.

FIG. 33 is an alignment of the amino acid sequences set forth in SEQ IDNOs:41, 46, 47, and 48.

FIG. 34 is a diagram of the construction of a synthetic operon (pTDH)that encodes for polypeptides having CoA transferase activity,lactyl-CoA dehydratase activity (E1, E2 α, and E2 β), and3-hydroxypropionyl-CoA dehydratase activity (3-HP-CoA dehydratase).

FIGS. 35A and B is a diagram of the construction of a synthetic operon(pHTD) that encodes for polypeptides having CoA transferase activity,lactyl-CoA dehydratase activity (E1, E2 α, and E2 β), and3-hydroxypropionyl-CoA dehydratase activity (3-HP-CoA dehydratase).

FIGS. 36A and B is a diagram of the construction of a synthetic operon(pEIITHrEI) that encodes for polypeptides having CoA transferaseactivity, lactyl-CoA dehydratase activity (E1, E2 α, and E2 β), and3-hydroxypropionyl-CoA dehydratase activity (3-HP-CoA dehydratase).

FIGS. 37A and B is a diagram of the construction of a synthetic operon(pEIITHEI) that encodes for polypeptides having CoA transferaseactivity, lactyl-CoA dehydratase activity (E1, E2 α, and E2 β), and3-hydroxypropionyl-CoA dehydratase activity (3-HP-CoA dehydratase).

FIGS. 38A and B is a diagram of the construction of two plasmids, pEIITHand pPROEI. The pEIITH plasmid encodes polypeptides having CoAtransferase activity, lactyl-CoA dehydratase activity (E2 α and E2 β),and 3-hydroxypropionyl-CoA dehydratase activity (3-HP-CoA dehydratase),and the pPROEI plasmid encodes a polypeptide having E1 activatoractivity.

FIG. 39 is a listing of a nucleic acid sequence that encodes apolypeptide having CoA synthase, dehydratase, and dehydrogenase activity(SEQ ID NO:129).

FIG. 40 is an alignment of the amino acid sequences set forth in SEQ IDNOs:39, 130, and 131. The uppercase amino acid residues representpositions where that amino acid residue is present in two or moresequences.

FIG. 41 is an alignment of the amino acid sequences set forth in SEQ IDNOs:39, 132, and 133. The uppercase amino acid residues representpositions where that amino acid residue is present in two or moresequences.

FIG. 42 is an alignment of the amino acid sequences set forth in SEQ IDNOs: 39, 134, and 135. The uppercase amino acid residues representpositions where that amino acid residue is present in two or moresequences.

FIG. 43 is a diagram of several pathways for making organic compoundsusing the multifunctional OS17 enzyme.

FIG. 44 is a diagram of a pathway for making 3-HP via acetyl-CoA andmalonyl-CoA.

FIG. 45 is a diagram of pMSD8, pET30a/acc1, pFN476, and PET286constructs.

FIG. 46 contains a total ion chromatogram and five mass spectrums ofCoenzyme A thioesters. Panel A is total ion chromatogram illustratingthe separation of Coenzyme A and four CoA-organic thioesters: 1=CoenzymeA, 2=lactyl-CoA, 3=acetyl-CoA, 4=acrylyl-CoA, 5=propionyl-CoA. Panel Bis a mass spectrum of Coenzyme A. Panel C is a mass spectrum oflactyl-CoA. Panel D is a mass spectrum of acetyl-CoA. Panel E is a massspectrum of acrylyl-CoA. Panel F is a mass spectrum of propionyl-CoA.

FIG. 47 contains ion chromatograms and mass spectrums. Panel A is atotal ion chromatogram of a mixture of lactyl-CoA and 3-HP-CoA. ThePanel A insert is the mass spectrum recorded under peak 1. Panel B is atotal ion chromatogram of lactyl-CoA. The Panel B insert is the massspectrum recorded under peak 2. In each panel, peak 1 is 3-HP-CoA, andpeak 2 is lactyl-CoA. The peak labeled with an asterisk was confirmednot to be a CoA ester.

FIG. 48 contains ion chromatograms and mass spectrums. Panel A is atotal ion chromatogram of CoA esters derived from a broth produced by E.coli transfected with pEIITHrEI. The Panel A insert is the mass spectrumrecorded under peak 1. Panel B is a total ion chromatogram of CoA estersderived from a broth produced by control E. coli not transfected withpEIITHrEI. The Panel B insert is the mass spectrum recorded under peak2. In each panel, peak 1 is 3-HP-CoA, and peak 2 is lactyl-CoA. Thepeaks labeled with an asterisk were confirmed not to be a CoA ester.

FIG. 49 is a listing of a nucleic acid sequence that encodes apolypeptide having malonyl-CoA reductase activity (SEQ ID NO: 140).

FIG. 50 is a listing of an amino acid sequence of a polypeptide havingmalonyl-CoA reductase activity (SEQ ID NO:141).

FIG. 51 is a listing of a nucleic acid sequence that encodes a portionof a polypeptide having malonyl-CoA reductase activity (SEQ ID NO:142).

FIG. 52 is an alignment of the amino acid sequences set forth in SEQ IDNOs: 141, 143, 144, 145, 146, and 147.

FIG. 53 is an alignment of the nucleic acid sequences set forth in SEQID NOs: 140, 148, 149, 150, 151, and 152.

FIG. 54 is a diagram of a pathway for making 3-HP via a β-alanineintermediate.

FIG. 55 is a diagram of a pathway for making 3-HP via a β-alanineintermediate.

FIG. 56 is a listing of an amino acid sequence of a polypeptide havingβ-alanyl-CoA ammonia lyase activity (SEQ ID NO:160).

FIG. 57 is a listing of an amino acid sequence of a polypeptide havingβ-alanyl-CoA ammonia lyase activity (SEQ ID NO:161).

FIG. 58 is a listing of a nucleic acid sequence that encodes apolypeptide having β-alanyl-CoA ammonia lyase activity (SEQ ID NO:162).

FIG. 59 is a listing of a nucleic acid sequence that can encode apolypeptide having β-alanyl-CoA ammonia lyase activity (SEQ ID NO:163).

DETAILED DESCRIPTION I. Terms

Nucleic acid: The term “nucleic acid” as used herein encompasses bothRNA and DNA including, without limitation, cDNA, genomic DNA, andsynthetic (e.g., chemically synthesized) DNA. The nucleic acid can bedouble-stranded or single-stranded. Where single-stranded, the nucleicacid can be the sense strand or the antisense strand. In addition,nucleic acid can be circular or linear.

Isolated: The term “isolated” as used herein with reference to nucleicacid refers to a naturally-occurring nucleic acid that is notimmediately contiguous with both of the sequences with which it isimmediately contiguous (one on the 5′ end and one on the 3′ end) in thenaturally-occurring genome of the organism from which it is derived. Forexample, an isolated nucleic acid can be, without limitation, arecombinant DNA molecule of any length, provided one of the nucleic acidsequences normally found immediately flanking that recombinant DNAmolecule in a naturally-occurring genome is removed or absent. Thus, anisolated nucleic acid includes, without limitation, a recombinant DNAthat exists as a separate molecule (e.g., a cDNA or a genomic DNAfragment produced by PCR or restriction endonuclease treatment)independent of other sequences as well as recombinant DNA that isincorporated into a vector, an autonomously replicating plasmid, a virus(e.g., a retrovirus, adenovirus, or herpes virus), or into the genomicDNA of a prokaryote or eukaryote. In addition, an isolated nucleic acidcan include a recombinant DNA molecule that is part of a hybrid orfusion nucleic acid sequence.

The term “isolated” as used herein with reference to nucleic acid alsoincludes any non-naturally-occurring nucleic acid sincenon-naturally-occurring nucleic acid sequences are not found in natureand do not have immediately contiguous sequences in anaturally-occurring genome. For example, non-naturally-occurring nucleicacid such as an engineered nucleic acid is considered to be isolatednucleic acid. Engineered nucleic acid can be made using common molecularcloning or chemical nucleic acid synthesis techniques. Isolatednon-naturally-occurring nucleic acid can be independent of othersequences, or incorporated into a vector, an autonomously replicatingplasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), orthe genomic DNA of a prokaryote or eukaryote. In addition, anon-naturally-occurring nucleic acid can include a nucleic acid moleculethat is part of a hybrid or fusion nucleic acid sequence.

It will be apparent to those of skill in the art that a nucleic acidexisting among hundreds to millions of other nucleic acid moleculeswithin, for example, cDNA or genomic libraries, or gel slices containinga genomic DNA restriction digest is not to be considered an isolatednucleic acid.

Exogenous: The term “exogenous” as used herein with reference to nucleicacid and a particular cell refers to any nucleic acid that does notoriginate from that particular cell as found in nature. Thus,non-naturally-occurring nucleic acid is considered to be exogenous to acell once introduced into the cell. Nucleic acid that isnaturally-occurring also can be exogenous to a particular cell. Forexample, an entire chromosome isolated from a cell of person X is anexogenous nucleic acid with respect to a cell of person Y once thatchromosome is introduced into Y's cell.

Hybridization: The term “hybridization” as used herein refers to amethod of testing for complementarity in the nucleotide sequence of twonucleic acid molecules, based on the ability of complementarysingle-stranded DNA and/or RNA to form a duplex molecule. Nucleic acidhybridization techniques can be used to obtain an isolated nucleic acidwithin the scope of the invention. Briefly, any nucleic acid having somehomology to a sequence set forth in SEQ ID NO:1, 9, 17, 25, 33, 34, 36,38, 40, 42, 129, 140, 142, 162, or 163 can be used as a probe toidentify a similar nucleic acid by hybridization under conditions ofmoderate to high stringency. Once identified, the nucleic acid then canbe purified, sequenced, and analyzed to determine whether it is withinthe scope of the invention as described herein.

Hybridization can be done by Southern or Northern analysis to identify aDNA or RNA sequence, respectively, that hybridizes to a probe. The probecan be labeled with a biotin, digoxygenin, an enzyme, or a radioisotopesuch as ³²P. The DNA or RNA to be analyzed can be electrophoreticallyseparated on an agarose or polyacrylamide gel, transferred tonitrocellulose, nylon, or other suitable membrane, and hybridized withthe probe using standard techniques well known in the art such as thosedescribed in sections 7.39-7.52 of Sambrook et al., (1989) MolecularCloning, second edition, Cold Spring Harbor Laboratory, Plainview, N.Y.Typically, a probe is at least about 20 nucleotides in length. Forexample, a probe corresponding to a 20 nucleotide sequence set forth inSEQ ID NO: 1, 9, 17, 25, 33, 34, 36, 38, 40, 42, 129, 140, or 142 can beused to identify an identical or similar nucleic acid. In addition,probes longer or shorter than 20 nucleotides can be used.

The invention also provides isolated nucleic acid sequences that are atleast about 12 bases in length (e.g., at least about 13, 14, 15, 16, 17,18, 19, 20, 25, 30, 40, 50, 60, 100, 250, 500, 750, 1000, 1500, 2000,3000, 4000, or 5000 bases in length) and hybridize, under hybridizationconditions, to the sense or antisense strand of a nucleic acid havingthe sequence set forth in SEQ ID NO:1, 9, 17, 25, 33, 34, 36, 38, 40,42, 129, 140, 142, 162, or 163. The hybridization conditions can bemoderately or highly stringent hybridization conditions.

For the purpose of this invention, moderately stringent hybridizationconditions mean the hybridization is performed at about 42° C. in ahybridization solution containing 25 mM KPO₄ (pH 7.4), 5×SSC,5×Denhart's solution, 50 μg/mL denatured, sonicated salmon sperm DNA,50% formamide, 10% Dextran sulfate, and 1-15 ng/mL probe (about 5×10⁷cpm/μg), while the washes are performed at about 50° C. with a washsolution containing 2×SSC and 0.1% sodium dodecyl sulfate.

Highly stringent hybridization conditions mean the hybridization isperformed at about 42° C. in a hybridization solution containing 25 mMKPO₄ (pH 7.4), 5×SSC, 5×Denhart's solution, 50 μg/mL denatured,sonicated salmon sperm DNA, 50% formamide, 10% Dextran sulfate, and 1-15ng/mL probe (about 5×10⁷ cpm/μg), while the washes are performed atabout 65° C. with a wash solution containing 0.2×SSC and 0.1% sodiumdodecyl sulfate.

Purified: The term “purified” as used herein does not require absolutepurity; rather, it is intended as a relative term. Thus, for example, apurified polypeptide or nucleic acid preparation can be one in which thesubject polypeptide or nucleic acid, respectively, is at a higherconcentration than the polypeptide or nucleic acid would be in itsnatural environment within an organism. For example, a polypeptidepreparation can be considered purified if the polypeptide content in thepreparation represents at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%,98%, or 99% of the total protein content of the preparation.

Transformed: A “transformed” cell is a cell into which a nucleic acidmolecule has been introduced by, for example, molecular biologytechniques. As used herein, the term “transformation” encompasses alltechniques by which a nucleic acid molecule might be introduced intosuch a cell including, without limitation, transfection with a viralvector, conjugation, transformation with a plasmid vector, andintroduction of naked DNA by electroporation, lipofection, and particlegun acceleration.

Recombinant: A “recombinant” nucleic acid is one having (1) a sequencethat is not naturally occurring in the organism in which it is expressedor (2) a sequence made by an artificial combination of twootherwise-separated, shorter sequences. This artificial combination isoften accomplished by chemical synthesis or, more commonly, by theartificial manipulation of isolated segments of nucleic acids, e.g., bygenetic engineering techniques. “Recombinant” is also used to describenucleic acid molecules that have been artificially manipulated, butcontain the same regulatory sequences and coding regions that are foundin the organism from which the nucleic acid was isolated.

Specific binding agent: A “specific binding agent” is an agent that iscapable of specifically binding to any of the polypeptide describedherein, and can include polyclonal antibodies, monoclonal antibodies(including humanized monoclonal antibodies), and fragments of monoclonalantibodies such as Fab, F(ab′)₂, and Fv fragments as well as any otheragent capable of specifically binding to an epitope of suchpolypeptides.

Antibodies to the polypeptides provided herein (or fragments thereof)can be used to purify or identify such polypeptides. The amino acid andnucleic acid sequences provided herein allow for the production ofspecific antibody-based binding agents that recognize the polypeptidesdescribed herein.

Monoclonal or polyclonal antibodies can be produced to the polypeptides,portions of the polypeptides, or variants thereof. Optimally, antibodiesraised against one or more epitopes on a polypeptide antigen willspecifically detect that polypeptide. That is, antibodies raised againstone particular polypeptide would recognize and bind that particularpolypeptide, and would not substantially recognize or bind to otherpolypeptides. The determination that an antibody specifically binds to aparticular polypeptide is made by any one of a number of standardimmunoassay methods; for instance, Western blotting (See, e.g., Sambrooket al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

To determine that a given antibody preparation (such as a preparationproduced in a mouse against a polypeptide having the amino acid sequenceset forth in SEQ ID NO: 2) specifically detects the appropriatepolypeptide (e.g., a polypeptide having the amino acid sequence setforth in SEQ ID NO: 2) by Western blotting, total cellular protein canbe extracted from cells and separated by SDS-polyacrylamide gelelectrophoresis. The separated total cellular protein can then betransferred to a membrane (e.g., nitrocellulose), and the antibodypreparation incubated with the membrane. After washing the membrane toremove non-specifically bound antibodies, the presence of specificallybound antibodies can be detected using an appropriate secondary antibody(e.g., an anti-mouse antibody) conjugated to an enzyme such as alkalinephosphatase since application of 5-bromo-4-chloro-3-indolylphosphate/nitro blue tetrazolium results in the production of a denselyblue-colored compound by immuno-localized alkaline phosphatase.

Substantially pure polypeptides suitable for use as an immunogen can beobtained from transfected cells, transformed cells, or wild-type cells.Polypeptide concentrations in the final preparation can be adjusted, forexample, by concentration on an Amicon filter device, to the level of afew micrograms per milliliter. In addition, polypeptides ranging in sizefrom full-length polypeptides to polypeptides having as few as nineamino acid residues can be utilized as immunogens. Such polypeptides canbe produced in cell culture, can be chemically synthesized usingstandard methods, or can be obtained by cleaving large polypeptides intosmaller polypeptides that can be purified. Polypeptides having as few asnine amino acid residues in length can be immunogenic when presented toan immune system in the context of a Major Histocompatibility Complex(MHC) molecule such as an MHC class I or MHC class II molecule.Accordingly, polypeptides having at least 9, 10, 11, 12, 13, 14, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 900, 1000, 1050, 1100,1150, 1200, 1250, 1300, 1350, or more consecutive amino acid residues ofany amino acid sequence disclosed herein can be used as immunogens forproducing antibodies.

Monoclonal antibodies to any of the polypeptides disclosed herein can beprepared from murine hybridomas according to the classic method ofKohler & Milstein (Nature 256:495 (1975)) or a derivative methodthereof.

Polyclonal antiserum containing antibodies to the heterogeneous epitopesof any polypeptide disclosed herein can be prepared by immunizingsuitable animals with the polypeptide (or fragment thereof), which canbe unmodified or modified to enhance immunogenicity. An effectiveimmunization protocol for rabbits can be found in Vaitukaitis et al. (J.Clin. Endocrinol. Metab. 33:988-991 (1971)).

Antibody fragments can be used in place of whole antibodies and can bereadily expressed in prokaryotic host cells. Methods of making and usingimmunologically effective portions of monoclonal antibodies, alsoreferred to as “antibody fragments,” are well known and include thosedescribed in Better & Horowitz (Methods Enzymol. 178:476-496 (1989)),Glockshuber et al. (Biochemistry 29:1362-1367 (1990), U.S. Pat. No.5,648,237 (“Expression of Functional Antibody Fragments”), U.S. Pat. No.4,946,778 (“Single Polypeptide Chain Binding Molecules”), U.S. Pat. No.5,455,030 (“Immunotherapy Using Single Chain Polypeptide BindingMolecules”), and references cited therein.

Operably linked: A first nucleic acid sequence is “operably linked” witha second nucleic acid sequence whenever the first nucleic acid sequenceis placed in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription of the codingsequence. Generally, operably linked DNA sequences are contiguous and,where necessary to join two polypeptide-coding regions, in the samereading frame.

Probes and primers: Nucleic acid probes and primers can be preparedreadily based on the amino acid sequences and nucleic acid sequencesprovided herein. A “probe” includes an isolated nucleic acid containinga detectable label or reporter molecule. Typical labels includeradioactive isotopes, ligands, chemiluminescent agents, and enzymes.Methods for labeling and guidance in the choice of labels appropriatefor various purposes are discussed in, for example, Sambrook et al.(ed.), Molecular Cloning: A Laboratory Manual 2nd ed., vol. 1-3, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, andAusubel et al. (ed.) Current Protocols in Molecular Biology, GreenePublishing and Wiley-Interscience, New York (with periodic updates),1987.

“Primers” are typically nucleic acid molecules having ten or morenucleotides (e.g., nucleic acid molecules having between about 10nucleotides and about 100 nucleotides). A primer can be annealed to acomplementary target nucleic acid strand by nucleic acid hybridizationto form a hybrid between the primer and the target nucleic acid strand,and then extended along the target nucleic acid strand by, for example,a DNA polymerase enzyme. Primer pairs can be used for amplification of anucleic acid sequence, for example, by the polymerase chain reaction(PCR) or other nucleic-acid amplification methods known in the art.

Methods for preparing and using probes and primers are described, forexample, in references such as Sambrook et al. (ed.), Molecular Cloning:A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989; Ausubel et al. (ed.), CurrentProtocols in Molecular Biology, Greene Publishing andWiley-Interscience, New York (with periodic updates), 1987; and Innis etal., PCR Protocols: A Guide to Methods and Applications, Academic Press:San Diego, 1990. PCR primer pairs can be derived from a known sequence,for example, by using computer programs intended for that purpose suchas Primer (Version 0.5, .COPYRGT. 1991, Whitehead Institute forBiomedical Research, Cambridge, Mass.). One of skill in the art willappreciate that the specificity of a particular probe or primerincreases with the length, but that a probe or primer can range in sizefrom a full-length sequence to sequences as short as five consecutivenucleotides. Thus, for example, a primer of 20 consecutive nucleotidescan anneal to a target with a higher specificity than a correspondingprimer of only 15 nucleotides. Thus, in order to obtain greaterspecificity, probes and primers can be selected that comprise, forexample, 10, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550,1600, 1650, 1700, 1750, 1800, 1850, 1900, 2000, 2050, 2100, 2150, 2200,2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800,2850, 2900, 3000, 3050, 3100, 3150, 3200, 3250, 3300, 3350, 3400, 3450,3500, 3550, 3600, 3650, 3700, 3750, 3800, 3850, 3900, 4000, 4050, 4100,4150, 4200, 4250, 4300, 4350, 4400, 4450, 4500, 4550, 4600, 4650, 4700,4750, 4800, 4850, 4900, 5000, 5050, 5100, 5150, 5200, 5250, 5300, 5350,5400, 5450, or more consecutive nucleotides.

Percent sequence identity: The “percent sequence identity” between aparticular nucleic acid or amino acid sequence and a sequence referencedby a particular sequence identification number is determined as follows.First, a nucleic acid or amino acid sequence is compared to the sequenceset forth in a particular sequence identification number using the BLAST2 Sequences (Bl2seq) program from the stand-alone version of BLASTZcontaining BLASTN version 2.0.14 and BLASTP version 2.0.14. Thisstand-alone version of BLASTZ can be obtained from Fish & Richardson'sweb site (www.fr.com) or the United States government's National Centerfor Biotechnology Information web site (www.ncbi.nlm.nih.gov).Instructions explaining how to use the Bl2seq program can be found inthe readme file accompanying BLASTZ. Bl2seq performs a comparisonbetween two sequences using either the BLASTN or BLASTP algorithm.BLASTN is used to compare nucleic acid sequences, while BLASTP is usedto compare amino acid sequences. To compare two nucleic acid sequences,the options are set as follows: −i is set to a file containing the firstnucleic acid sequence to be compared (e.g., C:\seq1.txt); −j is set to afile containing the second nucleic acid sequence to be compared (e.g.,C:\seq2.txt); −p is set to blastn; −o is set to any desired file name(e.g., C:\output.txt); −q is set to −1; −r is set to 2; and all otheroptions are left at their default setting. For example, the followingcommand can be used to generate an output file containing a comparisonbetween two sequences: C:\Bl2seq −i c:\seq1.txt −j c:\seq2.txt −p blastn−o c:\output.txt −q −1-r 2. To compare two amino acid sequences, theoptions of Bl2seq are set as follows: −i is set to a file containing thefirst amino acid sequence to be compared (e.g., C:\seq1.txt); −j is setto a file containing the second amino acid sequence to be compared(e.g., C:\seq2.txt); −p is set to blastp; −o is set to any desired filename (e.g., C:\output.txt); and all other options are left at theirdefault setting. For example, the following command can be used togenerate an output file containing a comparison between two amino acidsequences: C:\Bl2seq −i c:\seq1.txt −j c:\seq2.txt −p blastp −oc:\output.txt. If the two compared sequences share homology, then thedesignated output file will present those regions of homology as alignedsequences. If the two compared sequences do not share homology, then thedesignated output file will not present aligned sequences.

Once aligned, the number of matches is determined by counting the numberof positions where an identical nucleotide or amino acid residue ispresented in both sequences. The percent sequence identity is determinedby dividing the number of matches either by the length of the sequenceset forth in the identified sequence (e.g., SEQ ID NO:1), or by anarticulated length (e.g., 100 consecutive nucleotides or amino acidresidues from a sequence set forth in an identified sequence), followedby multiplying the resulting value by 100. For example, a nucleic acidsequence that has 1166 matches when aligned with the sequence set forthin SEQ ID NO:1 is 75.0 percent identical to the sequence set forth inSEQ ID NO:1 (i.e., 1166÷1554*100=75.0). It is noted that the percentsequence identity value is rounded to the nearest tenth. For example,75.11, 75.12, 75.13, and 75.14 is rounded down to 75.1, while 75.15,75.16, 75.17, 75.18, and 75.19 is rounded up to 75.2. It is also notedthat the length value will always be an integer. In another example, atarget sequence containing a 20-nucleotide region that aligns with 20consecutive nucleotides from an identified sequence as follows containsa region that shares 75 percent sequence identity to that identifiedsequence (i.e., 15÷20*100=75).

Conservative substitution: The term “conservative substitution” as usedherein refers to any of the amino acid substitutions set forth inTable 1. Typically, conservative substitutions have little to no impacton the activity of a polypeptide. A polypeptide can be produced tocontain one or more conservative substitutions by manipulating thenucleotide sequence that encodes that polypeptide using, for example,standard procedures such as site-directed mutagenesis or PCR.

TABLE 1 Original Conservative Residue Substitution(s) Ala ser Arg lysAsn gln; his Asp glu Cys ser Gln asn Glu asp Gly pro His asn; gln Ileleu; val Leu ile; val Lys arg; gln; glu Met leu; ile Phe met; leu; tyrSer thr Thr ser Trp tyr Tyr trp; phe Val ile; leu

II. Metabolic Pathways

The invention provides methods and materials related to producing 3-HPas well as other organic compounds (e.g., 1,3-propanediol, acrylic acid,polymerized acrylate, esters of acrylate, polymerized 3-HP, and estersof 3-HP). Specifically, the invention provides isolated nucleic acids,polypeptides, host cells, and methods and materials for producing 3-HPas well as other organic compounds such as 1,3-propanediol, acrylicacid, polymerized acrylate, esters of acrylate, polymerized 3-HP, andesters of 3-HP.

Accordingly, the invention provides several metabolic pathways that canbe used to produce organic compounds from PEP (FIGS. 1-5, 43-44, 54, and55). As depicted in FIG. 1, lactate can be converted into lactyl-CoA bya polypeptide having CoA transferase activity (EC 2.8.3.1); theresulting lactyl-CoA can be converted into acrylyl-CoA by a polypeptide(or multiple polypeptide complex such as an activated E2 α and E2 βcomplex) having lactyl-CoA dehydratase activity (EC 4.2.1.54); theresulting acrylyl-CoA can be converted into 3-hydroxypropionyl-CoA(3-HP-CoA) by a polypeptide having 3-hydroxypropionyl-CoA dehydrataseactivity (EC 4.2.1.-); and the resulting 3-HP-CoA can be converted into3-HP by a polypeptide having CoA transferase activity, a polypeptidehaving 3-hydroxypropionyl-CoA hydrolase activity (EC 3.1.2.-), or apolypeptide having 3-hydroxyisobutryl-CoA hydrolase activity (EC3.1.2.4).

Polypeptides having CoA transferase activity as well as nucleic acidencoding such polypeptides can be obtained from various speciesincluding, without limitation, Megasphaera elsdenii, Clostridiumpropionicum, Clostridium kluyveri, and Escherichia coli. For example,nucleic acid that encodes a polypeptide having CoA transferase activitycan be obtained from Megasphaera elsdenii as described in Example 1 andcan have a sequence as set forth in SEQ ID NO: 1. In addition,polypeptides having CoA transferase activity as well as nucleic acidencoding such polypeptides can be obtained as described herein. Forexample, the variations to SEQ ID NO: 1 provided herein can be used toencode a polypeptide having CoA transferase activity.

Polypeptides (or the polypeptides of a multiple polypeptide complex suchas an activated E2 α and E2 β complex) having lactyl-CoA dehydrataseactivity as well as nucleic acid encoding such polypeptides can beobtained from various species including, without limitation, Megasphaeraelsdenii and Clostridium propionicum. For example, nucleic acid encodingan E1 activator, an E2 α subunit, and an E2 β subunit that can form amultiple polypeptide complex having lactyl-CoA dehydratase activity canbe obtained from Megasphaera elsdenii as described in Example 2. Thenucleic acid encoding the E1 activator can contain a sequence as setforth in SEQ ID NO: 9; the nucleic acid encoding the E2 α subunit cancontain a sequence as set forth in SEQ ID NO: 17; and the nucleic acidencoding the E2 β subunit can contain a sequence as set forth in SEQ IDNO: 25. In addition, polypeptides (or the polypeptides of a multiplepolypeptide complex) having lactyl-CoA dehydratase activity as well asnucleic acid encoding such polypeptides can be obtained as describedherein. For example, the variations to SEQ ID NO: 9, 17, and 25 providedherein can be used to encode the polypeptides of a multiple polypeptidecomplex having CoA transferase activity.

Polypeptides having 3-hydroxypropionyl-CoA dehydratase activity as wellas nucleic acid encoding such polypeptides can be obtained from variousspecies including, without limitation, Chloroflexus aurantiacus, Candidarugosa, Rhodosprillium rubrum, and Rhodobacter capsulates. For example,nucleic acid that encodes a polypeptide having 3-hydroxypropionyl-CoAdehydratase activity can be obtained from Chloroflexus aurantiacus asdescribed in Example 3 and can have a sequence as set forth in SEQ IDNO: 40. In addition, polypeptides having 3-hydroxypropionyl-CoAdehydratase activity as well as nucleic acid encoding such polypeptidescan be obtained as described herein. For example, the variations to SEQID NO: 40 provided herein can be used to encode a polypeptide having3-hydroxypropionyl-CoA dehydratase activity.

Polypeptides having 3-hydroxypropionyl-CoA hydrolase activity as well asnucleic acid encoding such polypeptides can be obtained from variousspecies including, without limitation, Candida rugosa. Polypeptideshaving 3-hydroxyisobutryl-CoA hydrolase activity as well as nucleic acidencoding such polypeptides can be obtained from various speciesincluding, without limitation, Pseudomonas fluorescens, rattus, and homosapiens. For example, nucleic acid that encodes a polypeptide having3-hydroxyisobutryl-CoA hydrolase activity can be obtained from homosapiens and can have a sequence as set forth in GenBank® accessionnumber U66669.

The term “polypeptide having enzymatic activity” as used herein refersto any polypeptide that catalyzes a chemical reaction of othersubstances without itself being destroyed or altered upon completion ofthe reaction. Typically, a polypeptide having enzymatic activitycatalyzes the formation of one or more products from one or moresubstrates. Such polypeptides can have any type of enzymatic activityincluding, without limitation, the enzymatic activity or enzymaticactivities associated with enzymes such as dehydratases/hydratases,3-hydroxypropionyl-CoA dehydratases/hydratases, CoA transferases,lactyl-CoA dehydratases, 3-hydroxypropionyl-CoA hydrolases,3-hydroxyisobutryl-CoA hydrolases, poly hydroxyacid synthases, CoAsynthetases, malonyl-CoA reductases, β-alanine ammonia lyases, andlipases.

As depicted in FIG. 2, lactate can be converted into lactyl-CoA by apolypeptide having CoA synthetase activity (EC 6.2.1.-); the resultinglactyl-CoA can be converted into acrylyl-CoA by a polypeptide (ormultiple polypeptide complex) having lactyl-CoA dehydratase activity;the resulting acrylyl-CoA can be converted into 3-HP-CoA by apolypeptide having 3-hydroxypropionyl-CoA dehydratase activity; and theresulting 3-HP-CoA can be converted into polymerized 3-HP by apolypeptide having poly hydroxyacid synthase activity (EC 2.3.1.-).Polypeptides having CoA synthetase activity as well as nucleic acidencoding such polypeptides can be obtained from various speciesincluding, without limitation, Escherichia coli, Rhodobactersphaeroides, Saccharomyces cervisiae, and Salmonella enterica. Forexample, nucleic acid that encodes a polypeptide having CoA synthetaseactivity can be obtained from Escherichia coli and can have a sequenceas set forth in GenBank® accession number U00006. Polypeptides (ormultiple polypeptide complexes) having lactyl-CoA dehydratase activityas well as nucleic acid encoding such polypeptides can be obtained asprovided herein. Polypeptides having 3-hydroxypropionyl-CoA dehydrataseactivity as well as nucleic acid encoding such polypeptides also can beobtained as provided herein. Polypeptides having poly hydroxyacidsynthase activity as well as nucleic acid encoding such polypeptides canbe obtained from various species including, without limitation,Rhodobacter sphaeroides, Comamonas acidororans, Ralstonia eutropha, andPseudomonas oleovorans. For example, nucleic acid that encodes apolypeptide having poly hydroxyacid synthase activity can be obtainedfrom Rhodobacter sphaeroides and can have a sequence as set forth inGenBank® accession number X97200.

As depicted in FIG. 3, lactate can be converted into lactyl-CoA by apolypeptide having CoA transferase activity; the resulting lactyl-CoAcan be converted into acrylyl-CoA by a polypeptide (or multiplepolypeptide complex) having lactyl-CoA dehydratase activity; theresulting acrylyl-CoA can be converted into 3-HP-CoA by a polypeptidehaving 3-hydroxypropionyl-CoA dehydratase activity; the resulting3-HP-CoA can be converted into 3-HP by a polypeptide having CoAtransferase activity, a polypeptide having 3-hydroxypropionyl-CoAhydrolase activity, or a polypeptide having 3-hydroxyisobutryl-CoAhydrolase activity; and the resulting 3-HP can be converted into anester of 3-HP by a polypeptide having lipase activity (EC 3.1.1.-).Polypeptides having lipase activity as well as nucleic acid encodingsuch polypeptides can be obtained from various species including,without limitation, Candida rugosa, Candida tropicalis, and Candidaalbicans. For example, nucleic acid that encodes a polypeptide havinglipase activity can be obtained from Candida rugosa and can have asequence as set forth in GenBank® accession number A81171.

As depicted in FIG. 4, lactate can be converted into lactyl-CoA by apolypeptide having CoA synthetase activity; the resulting lactyl-CoA canbe converted into acrylyl-CoA by a polypeptide (or multiple polypeptidecomplex) having lactyl-CoA dehydratase activity; and the resultingacrylyl-CoA can be converted into polymerized acrylate by a polypeptidehaving poly hydroxyacid synthase activity.

As depicted in FIG. 5, lactate can be converted into lactyl-CoA by apolypeptide having CoA transferase activity; the resulting lactyl-CoAcan be converted into acrylyl-CoA by a polypeptide (or multiplepolypeptide complex) having lactyl-CoA dehydratase activity; theresulting acrylyl-CoA can be converted into acrylate by a polypeptidehaving CoA transferase activity; and the resulting acrylate can beconverted into an ester of acrylate by a polypeptide having lipaseactivity.

As depicted in FIG. 44, acetyl-CoA can be converted into malonyl-CoA bya polypeptide having acetyl-CoA carboxylase activity, and the resultingmalonyl-CoA can be converted into 3-HP by a polypeptide havingmalonyl-CoA reductase activity. Polypeptides having acetyl-CoAcarboxylase activity as well as nucleic acid encoding such polypeptidescan be obtained from various species including, without limitation,Escherichia coli and Chloroflexus aurantiacus. For example, nucleic acidthat encodes a polypeptide having acetyl-CoA carboxylase activity can beobtained from Escherichia coli and can have a sequence as set forth inGenBank® accession number M96394 or U18997. Polypeptides havingmalonyl-CoA reductase activity as well as nucleic acid encoding suchpolypeptides can be obtained from various species including, withoutlimitation, Chloroflexus aurantiacus, Sulfolobus metacillus, andAcidianus brierleyi. For example, nucleic acid that encodes apolypeptide having malonyl-CoA reductase activity can be obtained asdescribed herein and can have a sequence similar to the sequence setforth in SEQ ID NO: 140. In addition, polypeptides having malonyl-CoAreductase activity as well as nucleic acid encoding such polypeptidescan be obtained as described herein. For example, the variations to SEQID NO: 140 provided herein can be used to encode a polypeptide havingmalonyl-CoA reductase activity.

Polypeptides having malonyl-CoA reductase activity can use NADPH as aco-factor. For example, the polypeptide having the amino acid sequenceset forth in SEQ ID NO: 141 is a polypeptide having malonyl-CoAreductase activity that uses NADPH as a co-factor when convertingmalonyl-CoA into 3-HP. Likewise, polypeptides having malonyl-CoAreductase activity can use NADH as a co-factor. Such polypeptides can beobtained by converting a polypeptide that has malonyl-CoA reductaseactivity and uses NADPH as a cofactor into a polypeptide that hasmalonyl-CoA reductase activity and uses NADH as a cofactor. Any methodcan be used to convert a polypeptide that uses NADPH as a cofactor intoa polypeptide that uses NADH as a cofactor such as those described byothers (Eppink et al., J. Mol. Biol., 292(1):87-96 (1999), Hall andTomsett, Microbiology, 146(Pt 6):1399-406 (2000), and Dohr et al., Proc.Natl. Acad. Sci., 98(1):81-86 (2001)). For example, mutagenesis can beused to convert the polypeptide encoded by the nucleic acid sequence setforth in SEQ ID NO: 140 into a polypeptide that, when convertingmalonyl-CoA into 3-HP, uses NADH as a co-factor instead of NADPH.

As depicted in FIG. 43, propionate can be converted into propionyl-CoAby a polypeptide having CoA synthetase activity such as the polypeptidehaving the sequence set forth in SEQ ID NO: 39; the resultingpropionyl-CoA can be converted into acrylyl-CoA by a polypeptide havingdehydrogenase activity such as the polypeptide having the sequence setforth in SEQ ID NO: 39; and the resulting acrylyl-CoA can be convertedinto (1) acrylate by a polypeptide having CoA transferase activity orCoA hydrolase activity, (2) 3-HP-CoA by a polypeptide having 3-HPdehydratase activity (also referred to as acrylyl-CoA hydratase orsimply hydratase) such as the polypeptide having the sequence set forthin SEQ ID NO:39, or (3) polymerized acrylate by a polypeptide havingpoly hydroxyacid synthase activity. The resulting acrylate can beconverted into an ester of acrylate by a polypeptide having lipaseactivity. The resulting 3-HP-CoA can be converted into (1) 3-HP by apolypeptide having CoA transferase activity, a polypeptide having3-hydroxypropionyl-CoA hydrolase activity (EC 3.1.2.-), or a polypeptidehaving 3-hydroxyisobutyryl-CoA hydrolase activity (EC 3.1.2.4), or (2)polymerized 3-HP by a polypeptide having poly hydroxyacid synthaseactivity (EC 2.3.1.-).

As depicted in FIG. 54, PEP can be converted into β-alanine. β-alaninecan be converted into β-alanyl-CoA through the use of a polypeptidehaving CoA transferase activity. β-alanyl-CoA can then be converted intoacrylyl-CoA through the use of a polypeptide having β-alanyl-CoA ammonialyase activity. Acrylyl-CoA can then be converted into 3-HP-CoA throughthe use of a polypeptide having 3-HP-CoA dehydratase activity, and apolypeptide having glutamate dehydrogenase activity can be used toconvert 3-HP-CoA into 3-HP.

As depicted in FIG. 55, 3-HP can be made from β-alanine by firstcontacting β-alanine with a polypeptide having 4,4-aminobutyrateaminotransferase activity to create malonate semialdehyde. The malonatesemialdehyde can be converted into 3-HP with a polypeptide having 3-HPdehydrogenase activity or a polypeptide having 3-hydroxyisobutyratedehydrogenase activity.

III. Nucleic Acid Molecules and Polypeptides

The invention provides isolated nucleic acid that contains the entirenucleic acid sequence set forth in SEQ ID NO:1, 9, 17, 25, 33, 34, 36,38, 40, 42, 129, 140, 142, 162, or 163. In addition, the inventionprovides isolated nucleic acid that contains a portion of the nucleicacid sequence set forth in SEQ ID NO:1, 9, 17, 25, 33, 34, 36, 38, 40,42, 129, 140, 142, 162, or 163. For example, the invention providesisolated nucleic acid that contains a 15 nucleotide sequence identicalto any 15 nucleotide sequence set forth in SEQ ID NO:1, 9, 17, 25, 33,34, 36, 38, 40, 42, 129, 140, 142, 162, or 163 including, withoutlimitation, the sequence starting at nucleotide number 1 and ending atnucleotide number 15, the sequence starting at nucleotide number 2 andending at nucleotide number 16, the sequence starting at nucleotidenumber 3 and ending at nucleotide number 17, and so forth. It will beappreciated that the invention also provides isolated nucleic acid thatcontains a nucleotide sequence that is greater than 15 nucleotides(e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, ormore nucleotides) in length and identical to any portion of the sequenceset forth in SEQ ID NO:1, 9, 17, 25, 33, 34, 36, 38, 40, 42, 129, 140,142, 162, or 163. For example, the invention provides isolated nucleicacid that contains a 25 nucleotide sequence identical to any 25nucleotide sequence set forth in SEQ ID NO:1, 9, 17, 25, 33, 34, 36, 38,40, 42, 129, 140, 142, 162, or 163 including, without limitation, thesequence starting at nucleotide number 1 and ending at nucleotide number25, the sequence starting at nucleotide number 2 and ending atnucleotide number 26, the sequence starting at nucleotide number 3 andending at nucleotide number 27, and so forth. Additional examplesinclude, without limitation, isolated nucleic acids that contain anucleotide sequence that is 50 or more nucleotides (e.g., 100, 150, 200,250, 300, or more nucleotides) in length and identical to any portion ofthe sequence set forth in SEQ ID NO:1, 9, 17, 25, 33, 34, 36, 38, 40,42, 129, 140, 142, 162, or 163. Such isolated nucleic acids can include,without limitation, those isolated nucleic acids containing a nucleicacid sequence represented in a single line of sequence depicted in FIG.6, 10, 14, 18, 22, 23, 25, 27, 29, 31, 39, 49, or 51 since each line ofsequence depicted in these figures, with the possible exception of thelast line, provides a nucleotide sequence containing at least 50 bases.

In addition, the invention provides isolated nucleic acid that containsa variation of the nucleic acid sequence set forth in SEQ ID NO:1, 9,17, 25, 33, 34, 36, 38, 40, 42, 129, 140, 142, 162, or 163. For example,the invention provides isolated nucleic acid containing a nucleic acidsequence set forth in SEQ ID NO:1, 9, 17, 25, 33, 34, 36, 38, 40, 42,129, 140, 142, 162, or 163 that contains a single insertion, a singledeletion, a single substitution, multiple insertions, multipledeletions, multiple substitutions, or any combination thereof (e.g.,single deletion together with multiple insertions). Such isolatednucleic acid molecules can share at least 60, 65, 70, 75, 80, 85, 90,95, 97, 98, or 99 percent sequence identity with a sequence set forth inSEQ ID NO:1, 9, 17, 25, 33, 34, 36, 38, 40, 42, 129, 140, 142, 162, or163.

The invention provides multiple examples of isolated nucleic acid thatcontains a variation of a nucleic acid sequence set forth in SEQ IDNO:1, 9, 17, 25, 33, 34, 36, 38, 40, 42, 129, 140, 142, 162, or 163. Forexample, FIG. 8 provides the sequence set forth in SEQ ID NO:1 alignedwith three other nucleic acid sequences. Examples of variations of thesequence set forth in SEQ ID NO:1 include, without limitation, anyvariation of the sequence set forth in SEQ ID NO:1 provided in FIG. 8.Such variations are provided in FIG. 8 in that a comparison of thenucleotide (or lack thereof) at a particular position of the sequenceset forth in SEQ ID NO:1 with the nucleotide (or lack thereof) at thesame aligned position of any of the other three nucleic acid sequencesdepicted in FIG. 8 (i.e., SEQ ID NOs:3, 4, and 5) provides a list ofspecific changes for the sequence set forth in SEQ ID NO:1. For example,the “a” at position 49 of SEQ ID NO:1 can be substituted with an “c” asindicated in FIG. 8. As also indicated in FIG. 8, the “a” at position590 of SEQ ID NO:1 can be substituted with a “atgg”; an “aaac” can beinserted before the “g” at position 393 of SEQ ID NO:1; or the “gaa” atposition 736 of SEQ ID NO:1 can be deleted. It will be appreciated thatthe sequence set forth in SEQ ID NO:1 can contain any number ofvariations as well as any combination of types of variations. Forexample, the sequence set forth in SEQ ID NO:1 can contain one variationprovided in FIG. 8 or more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 50, 100, or more) of the variations provided in FIG. 8. Itis noted that the nucleic acid sequences provided by FIG. 8 can encodepolypeptides having CoA transferase activity. The invention alsoprovides isolated nucleic acid that contains a variant of a portion ofthe sequence set forth in SEQ ID NO:1 as depicted in FIG. 8 anddescribed herein.

Likewise, FIG. 12 provides variations of SEQ ID NO:9 and portionsthereof; FIG. 16 provides variations of SEQ ID NO:17 and portionsthereof; FIG. 20 provides variations of SEQ ID NO:25 and portionsthereof; FIG. 32 provides variations of SEQ ID NO:40 and portionsthereof; and FIG. 53 provides variations of SEQ ID NO:140.

The invention provides isolated nucleic acid that contains a nucleicacid sequence that encodes the entire amino acid sequence set forth inSEQ ID NO:2, 10, 18, 26, 35, 37, 39, 41, 141, 160, or 161. In addition,the invention provides isolated nucleic acid that contains a nucleicacid sequence that encodes a portion of the amino acid sequence setforth in SEQ ID NO:2, 10, 18, 26, 35, 37, 39, 41, 141, 160, or 161. Forexample, the invention provides isolated nucleic acid that contains anucleic acid sequence that encodes a 15 amino acid sequence identical toany 15 amino acid sequence set forth in SEQ ID NO:2, 10, 18, 26, 35, 37,39, 41, 141, 160, or 161 including, without limitation, the sequencestarting at amino acid residue number 1 and ending at amino acid residuenumber 15, the sequence starting at amino acid residue number 2 andending at amino acid residue number 16, the sequence starting at aminoacid residue number 3 and ending at amino acid residue number 17, and soforth. It will be appreciated that the invention also provides isolatednucleic acid that contains a nucleic acid sequence that encodes an aminoacid sequence that is greater than 15 amino acid residues (e.g., 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acidresidues) in length and identical to any portion of the sequence setforth in SEQ ID NO:2, 10, 18, 26, 35, 37, 39, 41, 141, 160, or 161. Forexample, the invention provides isolated nucleic acid that contains anucleic acid sequence that encodes a 25 amino acid sequence identical toany 25 amino acid sequence set forth in SEQ ID NO:2, 10, 18, 26, 35, 37,39, 41, 141, 160, or 161 including, without limitation, the sequencestarting at amino acid residue number 1 and ending at amino acid residuenumber 25, the sequence starting at amino acid residue number 2 andending at amino acid residue number 26, the sequence starting at aminoacid residue number 3 and ending at amino acid residue number 27, and soforth. Additional examples include, without limitation, isolated nucleicacids that contain a nucleic acid sequence that encodes an amino acidsequence that is 50 or more amino acid residues (e.g., 100, 150, 200,250, 300, or more amino acid residues) in length and identical to anyportion of the sequence set forth in SEQ ID NO:2, 10, 18, 26, 35, 37,39, 41, 141, 160, or 161. Such isolated nucleic acids can include,without limitation, those isolated nucleic acids containing a nucleicacid sequence that encodes an amino acid sequence represented in asingle line of sequence depicted in FIG. 7, 11, 15, 19, 24, 26, 28, 30,or 50 since each line of sequence depicted in these figures, with thepossible exception of the last line, provides an amino acid sequencecontaining at least 50 residues.

In addition, the invention provides isolated nucleic acid that containsa nucleic acid sequence that encodes an amino acid sequence having avariation of the amino acid sequence set forth in SEQ ID NO:2, 10, 18,26, 35, 37, 39, 41, 141, 160, or 161. For example, the inventionprovides isolated nucleic acid containing a nucleic acid sequenceencoding an amino acid sequence set forth in SEQ ID NO:2, 10, 18, 26,35, 37, 39, 41, 141, 160, or 161 that contains a single insertion, asingle deletion, a single substitution, multiple insertions, multipledeletions, multiple substitutions, or any combination thereof (e.g.,single deletion together with multiple insertions). Such isolatednucleic acid molecules can contain a nucleic acid sequence encoding anamino acid sequence that shares at least 60, 65, 70, 75, 80, 85, 90, 95,97, 98, or 99 percent sequence identity with a sequence set forth in SEQID NO:2, 10, 18, 26, 35, 37, 39, 41, 141, 160, or 161.

The invention provides multiple examples of isolated nucleic acidcontaining a nucleic acid sequence encoding an amino acid sequencehaving a variation of an amino acid sequence set forth in SEQ ID NO:2,10, 18, 26, 35, 37, 39, 41, 141, 160, or 161. For example, FIG. 9provides the amino acid sequence set forth in SEQ ID NO:2 aligned withthree other amino acid sequences. Examples of variations of the sequenceset forth in SEQ ID NO:2 include, without limitation, any variation ofthe sequence set forth in SEQ ID NO:2 provided in FIG. 9. Suchvariations are provided in FIG. 9 in that a comparison of the amino acidresidue (or lack thereof) at a particular position of the sequence setforth in SEQ ID NO:2 with the amino acid residue (or lack thereof) atthe same aligned position of any of the other three amino acid sequencesof FIG. 9 (i.e., SEQ ID NOs:6, 7, and 8) provides a list of specificchanges for the sequence set forth in SEQ ID NO:2. For example, the “k”at position 17 of SEQ ID NO:2 can be substituted with a “p” or “h” asindicated in FIG. 9. As also indicated in FIG. 9, the “v” at position125 of SEQ ID NO:2 can be substituted with an “i” or “f”. It will beappreciated that the sequence set forth in SEQ ID NO:2 can contain anynumber of variations as well as any combination of types of variations.For example, the sequence set forth in SEQ ID NO:2 can contain onevariation provided in FIG. 9 or more than one (e.g., 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 25, 50, 100, or more) of the variations provided inFIG. 9. It is noted that the amino acid sequences provided in FIG. 9 canbe polypeptides having CoA transferase activity.

The invention also provides isolated nucleic acid containing a nucleicacid sequence encoding an amino acid sequence that contains a variant ofa portion of the sequence set forth in SEQ ID NO:2 as depicted in FIG. 9and described herein.

Likewise, FIG. 13 provides variations of SEQ ID NO:10 and portionsthereof; FIG. 17 provides variations of SEQ ID NO:18 and portionsthereof; FIG. 21 provides variations of SEQ ID NO:26 and portionsthereof; FIG. 33 provides variations of SEQ ID NO:41 and portionsthereof; FIGS. 40, 41, and 42 provide variations of SEQ ID NO:39; andFIG. 52 provides variations of SEQ ID NO:141 and portions thereof.

It is noted that codon preferences and codon usage tables for aparticular species can be used to engineer isolated nucleic acidmolecules that take advantage of the codon usage preferences of thatparticular species. For example, the isolated nucleic acid providedherein can be designed to have codons that are preferentially used by aparticular organism of interest.

The invention also provides isolated nucleic acid that is at least about12 bases in length (e.g., at least about 13, 14, 15, 16, 17, 18, 19, 20,25, 30, 40, 50, 60, 100, 250, 500, 750, 1000, 1500, 2000, 3000, 4000, or5000 bases in length) and hybridizes, under hybridization conditions, tothe sense or antisense strand of a nucleic acid having the sequence setforth in SEQ ID NO:1, 9, 17, 25, 33, 34, 36, 38, 40, 42, 129, 140, 142,162, or 163. The hybridization conditions can be moderately or highlystringent hybridization conditions.

The invention provides polypeptides that contain the entire amino acidsequence set forth in SEQ ID NO:2, 10, 18, 26, 35, 37, 39, 41, 141, 160,or 161. In addition, the invention provides polypeptides that contain aportion of the amino acid sequence set forth in SEQ ID NO:2, 10, 18, 26,35, 37, 39, 41, 141, 160, or 161. For example, the invention providespolypeptides that contain a 15 amino acid sequence identical to any 15amino acid sequence set forth in SEQ ID NO:2, 10, 18, 26, 35, 37, 39,41, 141, 160, or 161 including, without limitation, the sequencestarting at amino acid residue number 1 and ending at amino acid residuenumber 15, the sequence starting at amino acid residue number 2 andending at amino acid residue number 16, the sequence starting at aminoacid residue number 3 and ending at amino acid residue number 17, and soforth. It will be appreciated that the invention also providespolypeptides that contain an amino acid sequence that is greater than 15amino acid residues (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, or more amino acid residues) in length and identical toany portion of the sequence set forth in SEQ ID NO:2, 10, 18, 26, 35,37, 39, 41, 141, 160, or 161. For example, the invention providespolypeptides that contain a 25 amino acid sequence identical to any 25amino acid sequence set forth in SEQ ID NO:2, 10, 18, 26, 35, 37, 39,41, 141, 160, or 161 including, without limitation, the sequencestarting at amino acid residue number 1 and ending at amino acid residuenumber 25, the sequence starting at amino acid residue number 2 andending at amino acid residue number 26, the sequence starting at aminoacid residue number 3 and ending at amino acid residue number 27, and soforth. Additional examples include, without limitation, polypeptidesthat contain an amino acid sequence that is 50 or more amino acidresidues (e.g., 100, 150, 200, 250, 300, or more amino acid residues) inlength and identical to any portion of the sequence set forth in SEQ IDNO:2, 10, 18, 26, 35, 37, 39, 41, 141, 160, or 161. Such polypeptidescan include, without limitation, those polypeptides containing a aminoacid sequence represented in a single line of sequence depicted in FIG.7, 11, 15, 19, 24, 26, 28, 30, or 50 since each line of sequencedepicted in these figures, with the possible exception of the last line,provides an amino acid sequence containing at least 50 residues.

In addition, the invention provides polypeptides that an amino acidsequence having a variation of the amino acid sequence set forth in SEQID NO:2, 10, 18, 26, 35, 37, 39, 41, 141, 160, or 161. For example, theinvention provides polypeptides containing an amino acid sequence setforth in SEQ ID NO:2, 10, 18, 26, 35, 37, 39, 41, 141, 160, or 161 thatcontains a single insertion, a single deletion, a single substitution,multiple insertions, multiple deletions, multiple substitutions, or anycombination thereof (e.g., single deletion together with multipleinsertions). Such polypeptides can contain an amino acid sequence thatshares at least 60, 65, 70, 75, 80, 85, 90, 95, 97, 98, or 99 percentsequence identity with a sequence set forth in SEQ ID NO:2, 10, 18, 26,35, 37, 39, 41, 141, 160, or 161.

The invention provides multiple examples of polypeptides containing anamino acid sequence having a variation of an amino acid sequence setforth in SEQ ID NO:2, 10, 18, 26, 35, 37, 39, 41, 141, 160, or 161. Forexample, FIG. 9 provides the amino acid sequence set forth in SEQ IDNO:2 aligned with three other amino acid sequences. Examples ofvariations of the sequence set forth in SEQ ID NO:2 include, withoutlimitation, any variation of the sequence set forth in SEQ ID NO:2provided in FIG. 9. Such variations are provided in FIG. 9 in that acomparison of the amino acid residue (or lack thereof) at a particularposition of the sequence set forth in SEQ ID NO:2 with the amino acidresidue (or lack thereof) at the same aligned position of any of theother three amino acid sequences of FIG. 9 (i.e., SEQ ID NOs:6, 7, and8) provides a list of specific changes for the sequence set forth in SEQID NO:2. For example, the “k” at position 17 of SEQ ID NO:2 can besubstituted with a “p” or “h” as indicated in FIG. 9. As also indicatedin FIG. 9, the “v” at position 125 of SEQ ID NO:2 can be substitutedwith an “i” or “f”. It will be appreciated that the sequence set forthin SEQ ID NO:2 can contain any number of variations as well as anycombination of types of variations. For example, the sequence set forthin SEQ ID NO:2 can contain one variation provided in FIG. 9 or more thanone (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, or more) ofthe variations provided in FIG. 9. It is noted that the amino acidsequences provided in FIG. 9 can be polypeptides having CoA transferaseactivity.

The invention also provides polypeptides containing an amino acidsequence that contains a variant of a portion of the sequence set forthin SEQ ID NO:2 as depicted in FIG. 9 and described herein.

Likewise, FIG. 13 provides variations of SEQ ID NO:10 and portionsthereof; FIG. 17 provides variations of SEQ ID NO:18 and portionsthereof; FIG. 21 provides variations of SEQ ID NO:26 and portionsthereof; FIG. 33 provides variations of SEQ ID NO:41 and portionsthereof, FIGS. 40, 41, and 42 provide variations of SEQ ID NO:39; andFIG. 52 provides variations of SEQ ID NO:141 and portions thereof.

Polypeptides having a variant amino acid sequence can retain enzymaticactivity. Such polypeptides can be produced by manipulating thenucleotide sequence encoding a polypeptide using standard proceduressuch as site-directed mutagenesis or PCR. One type of modificationincludes the substitution of one or more amino acid residues for aminoacid residues having a similar biochemical property. For example, apolypeptide can have an amino acid sequence set forth in SEQ ID NO:2,10, 18, 26, 35, 37, 39, 41, 141, 160, or 161 with one or moreconservative substitutions.

More substantial changes can be obtained by selecting substitutions thatare less conservative than those in Table 1, i.e., selecting residuesthat differ more significantly in their effect on maintaining: (a) thestructure of the polypeptide backbone in the area of the substitution,for example, as a sheet or helical conformation; (b) the charge orhydrophobicity of the polypeptide at the target site; or (c) the bulk ofthe side chain. The substitutions that in general are expected toproduce the greatest changes in polypeptide function are those in which:(a) a hydrophilic residue, e.g., serine or threonine, is substituted for(or by) a hydrophobic residue, e.g., leucine, isoleucine, phenylalanine,valine or alanine; (b) a cysteine or proline is substituted for (or by)any other residue; (c) a residue having an electropositive side chain,e.g., lysine, arginine, or histidine, is substituted for (or by) anelectronegative residue, e.g., glutamic acid or aspartic acid; or (d) aresidue having a bulky side chain, e.g., phenylalanine, is substitutedfor (or by) one not having a side chain, e.g., glycine. The effects ofthese amino acid substitutions (or other deletions or additions) can beassessed for polypeptides having enzymatic activity by analyzing theability of the polypeptide to catalyze the conversion of the samesubstrate as the related native polypeptide to the same product as therelated native polypeptide. Accordingly, polypeptides having 5, 10, 20,30, 40, 50 or less conservative substitutions are provided by theinvention.

Polypeptides and nucleic acid encoding polypeptide can be produced bystandard DNA mutagenesis techniques, for example, M13 primermutagenesis. Details of these techniques are provided in Sambrook et al.(ed.), Molecular Cloning: A Laboratory Manual 2nd ed., vol. 1-3, ColdSpring Harbor Laboratory Press, Cold Spring, Harbor, N.Y., 1989, Ch. 15.Nucleic acid molecules can contain changes of a coding region to fit thecodon usage bias of the particular organism into which the molecule isto be introduced.

Alternatively, the coding region can be altered by taking advantage ofthe degeneracy of the genetic code to alter the coding sequence in sucha way that, while the nucleic acid sequence is substantially altered, itnevertheless encodes a polypeptide having an amino acid sequenceidentical or substantially similar to the native amino acid sequence.For example, the ninth amino acid residue of the sequence set forth inSEQ ID NO: 2 is alanine, which is encoded in the open reading frame bythe nucleotide codon triplet GCT. Because of the degeneracy of thegenetic code, three other nucleotide codon triplets—GCA, GCC, andGCG—also code for alanine. Thus, the nucleic acid sequence of the openreading frame can be changed at this position to any of these threecodons without affecting the amino acid sequence of the encodedpolypeptide or the characteristics of the polypeptide. Based upon thedegeneracy of the genetic code, nucleic acid variants can be derivedfrom a nucleic acid sequence disclosed herein using a standard DNAmutagenesis techniques as described herein, or by synthesis of nucleicacid sequences. Thus, this invention also encompasses nucleic acidmolecules that encode the same polypeptide but vary in nucleic acidsequence by virtue of the degeneracy of the genetic code.

IV. Methods of Making 3-HP and Other Organic Acids

Each step provided in the pathways depicted in FIGS. 1-5, 43-44, 54, and55 can be performed within a cell (in vivo) or outside a cell (in vitro,e.g., in a container or column). Additionally, the organic acid productscan be generated through a combination of in vivo synthesis and in vitrosynthesis. Moreover, the in vitro synthesis step, or steps, can be viachemical reaction or enzymatic reaction.

For example, a microorganism provided herein can be used to perform thesteps provided in FIG. 1, or an extract containing polypeptides havingthe indicated enzymatic activities can be used to perform the stepsprovided in FIG. 1. In addition, chemical treatments can be used toperform the conversions provided in FIGS. 1-5, 43-44, 54, and 55. Forexample, acrylyl-CoA can be converted into acrylate by hydrolysis. Otherchemical treatments include, without limitation, trans esterification toconvert acrylate into an acrylate ester.

Carbon sources suitable as starting points for bioconversion includecarbohydrates and synthetic intermediates. Examples of carbohydrateswhich cells are capable of metabolizing to pyruvate include sugars suchas dextrose, triglycerides, and fatty acids.

Additionally, intermediate chemical products can be starting points. Forexample, acetic acid and carbon dioxide can be introduced into afermentation broth. Acetyl-CoA, malonyl-CoA, and 3-HP can besequentially produced using a polypeptide having CoA synthase activity,a polypeptide having acetyl-CoA carboxylase activity, and a polypeptidehaving malonyl-CoA reductase activity. Other useful intermediatechemical starting points can include propionic acid, acrylic acid,lactic acid, pyruvic acid, and β-alanine.

A. Expression of Polypeptides

The polypeptides described herein can be produced individually in a hostcell or in combination in a host cell. Moreover, the polypeptides havinga particular enzymatic activity can be a polypeptide that is eithernaturally-occurring or non-naturally-occurring. A naturally-occurringpolypeptide is any polypeptide having an amino acid sequence as found innature, including wild-type and polymorphic polypeptides. Suchnaturally-occurring polypeptides can be obtained from any speciesincluding, without limitation, animal (e.g., mammalian), plant, fungal,and bacterial species. A non-naturally-occurring polypeptide is anypolypeptide having an amino acid sequence that is not found in nature.Thus, a non-naturally-occurring polypeptide can be a mutated version ofa naturally-occurring polypeptide, or an engineered polypeptide. Forexample, a non-naturally-occurring polypeptide having3-hydroxypropionyl-CoA dehydratase activity can be a mutated version ofa naturally-occurring polypeptide having 3-hydroxypropionyl-CoAdehydratase activity that retains at least some 3-hydroxypropionyl-CoAdehydratase activity. A polypeptide can be mutated by, for example,sequence additions, deletions, substitutions, or combinations thereof.

The invention provides genetically modified cells that can be used toperform one or more steps of the steps in the metabolic pathwaysdescribed herein or the genetically modified cells can be used toproduce the disclosed polypeptides for subsequent use in vitro. Forexample, an individual microorganism can contain exogenous nucleic acidsuch that each of the polypeptides necessary to perform the stepsdepicted in FIG. 1, 2, 3, 4, 5, 43, 44, 54, or 55 are expressed. It isimportant to note that such cells can contain any number of exogenousnucleic acid molecules. For example, a particular cell can contain sixexogenous nucleic acid molecules with each one encoding one of the sixpolypeptides necessary to convert lactate into 3-HP as depicted in FIG.1, or a particular cell can endogenously produce polypeptides necessaryto convert lactate into acrylyl-CoA while containing exogenous nucleicacid that encodes polypeptides necessary to convert acrylyl-CoA into3-HP.

In addition, a single exogenous nucleic acid molecule can encode one ormore than one polypeptide. For example, a single exogenous nucleic acidmolecule can contain sequences that encode three different polypeptides.Further, the cells described herein can contain a single copy, ormultiple copies (e.g., about 5, 10, 20, 35, 50, 75, 100 or 150 copies),of a particular exogenous nucleic acid molecule. For example, aparticular cell can contain about 50 copies of the constructs depictedin FIG. 34, 35, 36, 37, 38, or 45. Again, the cells described herein cancontain more than one particular exogenous nucleic acid molecule. Forexample, a particular cell can contain about 50 copies of exogenousnucleic acid molecule X as well as about 75 copies of exogenous nucleicacid molecule Y.

In another embodiment, a cell within the scope of the invention cancontain an exogenous nucleic acid molecule that encodes a polypeptidehaving 3-hydroxypropionyl-CoA dehydratase activity. Such cells can haveany level of 3-hydroxypropionyl-CoA dehydratase activity. For example, acell containing an exogenous nucleic acid molecule that encodes apolypeptide having 3-hydroxypropionyl-CoA dehydratase activity can have3-hydroxypropionyl-CoA dehydratase activity with a specific activitygreater than about 1 mg 3-HP-CoA formed per gram dry cell weight perhour (e.g., greater than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,125, 150, 200, 250, 300, 350, 400, 500, or more mg 3-HP-CoA formed pergram dry cell weight per hour). Alternatively, a cell can have3-hydroxypropionyl-CoA dehydratase activity such that a cell extractfrom 1×10⁶ cells has a specific activity greater than about 1 μg3-HP-CoA formed per mg total protein per 10 minutes (e.g., greater thanabout 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 250, 300,350, 400, 500, or more μg 3-HP-CoA formed per mg total protein per 10minutes).

A nucleic acid molecule encoding a polypeptide having enzymatic activitycan be identified and obtained using any method such as those describedherein. For example, nucleic acid molecules that encode a polypeptidehaving enzymatic activity can be identified and obtained using commonmolecular cloning or chemical nucleic acid synthesis procedures andtechniques, including PCR. In addition, standard nucleic acid sequencingtechniques and software programs that translate nucleic acid sequencesinto amino acid sequences based on the genetic code can be used todetermine whether or not a particular nucleic acid has any sequencehomology with known enzymatic polypeptides. Sequence alignment softwaresuch as MEGALIGN® (DNASTAR, Madison, Wis., 1997) can be used to comparevarious sequences. In addition, nucleic acid molecules encoding knownenzymatic polypeptides can be mutated using common molecular cloningtechniques (e.g., site-directed mutagenesis). Possible mutationsinclude, without limitation, deletions, insertions, and basesubstitutions, as well as combinations of deletions, insertions, andbase substitutions. Further, nucleic acid and amino acid databases(e.g., GenBank®) can be used to identify a nucleic acid sequence thatencodes a polypeptide having enzymatic activity. Briefly, any amino acidsequence having some homology to a polypeptide having enzymaticactivity, or any nucleic acid sequence having some homology to asequence encoding a polypeptide having enzymatic activity can be used asa query to search GenBank®. The identified polypeptides then can beanalyzed to determine whether or not they exhibit enzymatic activity.

In addition, nucleic acid hybridization techniques can be used toidentify and obtain a nucleic acid molecule that encodes a polypeptidehaving enzymatic activity. Briefly, any nucleic acid molecule thatencodes a known enzymatic polypeptide, or fragment thereof, can be usedas a probe to identify a similar nucleic acid molecules by hybridizationunder conditions of moderate to high stringency. Such similar nucleicacid molecules then can be isolated, sequenced, and analyzed todetermine whether the encoded polypeptide has enzymatic activity.

Expression cloning techniques also can be used to identify and obtain anucleic acid molecule that encodes a polypeptide having enzymaticactivity. For example, a substrate known to interact with a particularenzymatic polypeptide can be used to screen a phage display librarycontaining that enzymatic polypeptide. Phage display libraries can begenerated as described elsewhere (Burritt et al., Anal. Biochem.238:1-13 (1990)), or can be obtained from commercial suppliers such asNovagen (Madison, Wis.).

Further, polypeptide sequencing techniques can be used to identify andobtain a nucleic acid molecule that encodes a polypeptide havingenzymatic activity. For example, a purified polypeptide can be separatedby gel electrophoresis, and its amino acid sequence determined by, forexample, amino acid microsequencing techniques. Once determined, theamino acid sequence can be used to design degenerate oligonucleotideprimers. Degenerate oligonucleotide primers can be used to obtain thenucleic acid encoding the polypeptide by PCR. Once obtained, the nucleicacid can be sequenced, cloned into an appropriate expression vector, andintroduced into a microorganism.

Any method can be used to introduce an exogenous nucleic acid moleculeinto a cell. In fact, many methods for introducing nucleic acid intomicroorganisms such as bacteria and yeast are well known to thoseskilled in the art. For example, heat shock, lipofection,electroporation, conjugation, fusion of protoplasts, and biolisticdelivery are common methods for introducing nucleic acid into bacteriaand yeast cells. See, e.g., Ito et al., J. Bacterol. 153:163-168 (1983);Durrens et al., Curr. Genet. 18:7-12 (1990); and Becker and Guarente,Methods in Enzymology 194:182-187 (1991).

An exogenous nucleic acid molecule contained within a particular cell ofthe invention can be maintained within that cell in any form. Forexample, exogenous nucleic acid molecules can be integrated into thegenome of the cell or maintained in an episomal state. In other words, acell of the invention can be a stable or transient transformant. Again,a microorganism described herein can contain a single copy, or multiplecopies (e.g., about 5, 10, 20, 35, 50, 75, 100 or 150 copies), of aparticular exogenous nucleic acid molecule as described herein.

Methods for expressing an amino acid sequence from an exogenous nucleicacid molecule are well known to those skilled in the art. Such methodsinclude, without limitation, constructing a nucleic acid such that aregulatory element promotes the expression of a nucleic acid sequencethat encodes a polypeptide. Typically, regulatory elements are DNAsequences that regulate the expression of other DNA sequences at thelevel of transcription. Thus, regulatory elements include, withoutlimitation, promoters, enhancers, and the like. Any type of promoter canbe used to express an amino acid sequence from an exogenous nucleic acidmolecule. Examples of promoters include, without limitation,constitutive promoters, tissue-specific promoters, and promotersresponsive or unresponsive to a particular stimulus (e.g., light,oxygen, chemical concentration, and the like). Moreover, methods forexpressing a polypeptide from an exogenous nucleic acid molecule incells such as bacterial cells and yeast cells are well known to thoseskilled in the art. For example, nucleic acid constructs that arecapable of expressing exogenous polypeptides within E. coli are wellknown. See, e.g., Sambrook et al., Molecular cloning: a laboratorymanual, Cold Spring Harbour Laboratory Press, New York, USA, secondedition (1989).

B. Production of Organic Acids and Related Products Via Host Cells

The nucleic acid and amino acid sequences provided herein can be usedwith cells to produce 3-HP and/or other organic compounds such as1,3-propanediol, acrylic acid, polymerized acrylate, esters of acrylate,esters of 3-HP, and polymerized 3-HP. Such cells can be from any speciesincluding those listed within the taxonomy web pages at the NationalInstitute of Health sponsored by the United States government(www.ncbi.nlm.nih.gov). The cells can be eukaryotic or prokaryotic. Forexample, genetically modified cells of the invention can be mammaliancells (e.g., human, murine, and bovine cells), plant cells (e.g., corn,wheat, rice, and soybean cells), fungal cells (e.g., Aspergillus andRhizopus cells), yeast cells, or bacterial cells (e.g., Lactobacillus,Lactococcus, Bacillus, Escherichia, and Clostridium cells). A cell ofthe invention also can be a microorganism. The term “microorganism” asused herein refers to any microscopic organism including, withoutlimitation, bacteria, algae, fungi, and protozoa. Thus, E. coli, S.cerevisiae, Kluveromyces lactis, Candida blankii, Candida rugosa, andPichia postoris are considered microorganisms and can be used asdescribed herein.

Typically, a cell of the invention is genetically modified such that aparticular organic compound is produced. In one embodiment, theinvention provides cells that make 3-HP from PEP. Examples biosyntheticpathways that may be used by cells to make 3-HP are shown in FIGS. 1-5,43-44, 54, and 55.

Generally, cells that are genetically modified to synthesize aparticular organic compound contain one or more exogenous nucleic acidmolecules that encode polypeptides having specific enzymatic activities.For example, a microorganism can contain exogenous nucleic acid thatencodes a polypeptide having 3-hydroxypropionyl-CoA dehydrataseactivity. In this case, acrylyl-CoA can be converted into3-hydroxypropionic acid-CoA which can lead to the production of 3-HP. Itis noted that a cell can be given an exogenous nucleic acid moleculethat encodes a polypeptide having an enzymatic activity that catalyzesthe production of a compound not normally produced by that cell.Alternatively, a cell can be given an exogenous nucleic acid moleculethat encodes a polypeptide having an enzymatic activity that catalyzesthe production of a compound that is normally produced by that cell. Inthis case, the genetically modified cell can produce more of thecompound, or can produce the compound more efficiently, than a similarcell not having the genetic modification.

In one embodiment, the invention provides a cell containing an exogenousnucleic acid molecule that encodes a polypeptide having enzymaticactivity that leads to the formation of 3-HP. It is noted that theproduced 3-HP can be secreted from the cell, eliminating the need todisrupt cell membranes to retrieve the organic compound. Typically, thecell of the invention produces 3-HP with the concentration being atleast about 100 mg per L (e.g., at least about 1 g/L, 5 g/L, 10 g/L, 25g/L, 50 g/L, 75 g/L, 80 g/L, 90 g/L, 100 g/L, or 120 g/L). Whendetermining the yield of an organic compound such as 3-HP for aparticular cell, any method can be used. See, e.g., AppliedEnvironmental Microbiology 59(12):4261-4265 (1993). Typically, a cellwithin the scope of the invention such as a microorganism catabolizes ahexose carbon source such as glucose. A cell, however, can catabolize avariety of carbon sources such as pentose sugars (e.g., ribose,arabinose, xylose, and lyxose), fatty acids, acetate, or glycerols. Inother words, a cell within the scope of the invention can utilize avariety of carbon sources.

As described herein, a cell within the scope of the invention cancontain an exogenous nucleic acid molecule that encodes a polypeptidehaving enzymatic activity that leads to the formation of 3-HP or otherorganic compounds such as 1,3-propanediol, acrylic acid, poly-acrylate,acrylate-esters, 3-HP-esters, and poly-3-HP. Methods of identifyingcells that contain exogenous nucleic acid are well known to thoseskilled in the art. Such methods include, without limitation, PCR andnucleic acid hybridization techniques such as Northern and Southernanalysis (see hybridization described herein). In some cases,immunohisto-chemistry and biochemical techniques can be used todetermine if a cell contains a particular nucleic acid by detecting theexpression of the polypeptide encoded by that particular nucleic acidmolecule. For example, an antibody having specificity for a polypeptidecan be used to determine whether or not a particular cell containsnucleic acid encoding that polypeptide. Further, biochemical techniquescan be used to determine if a cell contains a particular nucleic acidmolecule encoding a polypeptide having enzymatic activity by detectingan organic product produced as a result of the expression of thepolypeptide having enzymatic activity. For example, detection of 3-HPafter introduction of exogenous nucleic acid that encodes a polypeptidehaving 3-hydroxypropionyl-CoA dehydratase activity into a cell that doesnot normally express such a polypeptide can indicate that that cell notonly contains the introduced exogenous nucleic acid molecule but alsoexpresses the encoded polypeptide from that introduced exogenous nucleicacid molecule. Methods for detecting specific enzymatic activities orthe presence of particular organic products are well known to thoseskilled in the art. For example, the presence of an organic compoundsuch as 3-HP can be determined as described elsewhere. See, Sullivan andClarke, J. Assoc. Offic. Agr. Chemists, 38:514-518 (1955).

C. Cells with Reduced Polypeptide Activity

The invention also provides genetically modified cells having reducedpolypeptide activity. The term “reduced” as used herein with respect toa cell and a particular polypeptide's activity refers to a lower levelof activity than that measured in a comparable cell of the same species.For example, a particular microorganism lacking enzymatic activity X isconsidered to have reduced enzymatic activity X if a comparablemicroorganism has at least some enzymatic activity X. It is noted that acell can have the activity of any type of polypeptide reduced including,without limitation, enzymes, transcription factors, transporters,receptors, signal molecules, and the like. For example, a cell cancontain an exogenous nucleic acid molecule that disrupts a regulatoryand/or coding sequence of a polypeptide having pyruvate decarboxylaseactivity or alcohol dehydrogenase activity. Disrupting pyruvatedecarboxylase and/or alcohol dehydrogenase expression can lead to theaccumulation of lactate as well as products produced from lactate suchas 3-HP, 1,3-propanediol, acrylic acid, poly-acrylate, acrylate-esters,3-HP-esters, and poly-3-HP. It is also noted that reduced polypeptideactivities can be the result of lower polypeptide concentration, lowerspecific activity of a polypeptide, or combinations thereof. Manydifferent methods can be used to make a cell having reduced polypeptideactivity. For example, a cell can be engineered to have a disruptedregulatory sequence or polypeptide-encoding sequence using commonmutagenesis or knock-out technology. See, e.g., Methods in YeastGenetics (1997 edition), Adams, Gottschling, Kaiser, and Stems, ColdSpring Harbor Press (1998). Alternatively, antisense technology can beused to reduce the activity of a particular polypeptide. For example, acell can be engineered to contain a cDNA that encodes an antisensemolecule that prevents a polypeptide from being translated. The term“antisense molecule” as used herein encompasses any nucleic acidmolecule or nucleic acid analog (e.g., peptide nucleic acids) thatcontains a sequence that corresponds to the coding strand of anendogenous polypeptide. An antisense molecule also can have flankingsequences (e.g., regulatory sequences). Thus, antisense molecules can beribozymes or antisense oligonucleotides. A ribozyme can have any generalstructure including, without limitation, hairpin, hammerhead, or axheadstructures, provided the molecule cleaves RNA. Further, gene silencingcan be used to reduce the activity of a particular polypeptide.

A cell having reduced activity of a polypeptide can be identified usingany method. For example, enzyme activity assays such as those describedherein can be used to identify cells having a reduced enzyme activity.

A polypeptide having (1) the amino acid sequence set forth in SEQ IDNO:39 (the OS17 polypeptide) or (2) an amino acid sequence sharing atleast about 60 percent sequence identity with the amino acid sequenceset forth in SEQ ID NO:39 can have three functional domains: a domainhaving CoA-synthatase activity, a domain having 3-HP-CoA dehydrataseactivity, and a domain having CoA-reductase activity. Such polypeptidescan be selectively modified by mutating and/or deleting domains suchthat one or two of the enzymatic activities are reduced. Reducing thedehydratase activity of the OS17 polypeptide can cause acrylyl-CoA to becreated from propionyl-CoA. The acrylyl-CoA then can be contacted with apolypeptide having CoA hydrolase activity to produce acrylate frompropionate (FIG. 43). Similarly, acrylyl-CoA can be created from 3-HP byusing, for example, an OS17 polypeptide having reduced reductaseactivity.

D. Production of Organic Acids and Related Products Via In VitroTechniques

In addition, purified polypeptides having enzymatic activity can be usedalone or in combination with cells to produce 3-HP or other organiccompounds such as 1,3-propanediol, acrylic acid, polymerized acrylate,esters of acrylate, esters of 3-HP, and polymerized 3-HP. For example, apreparation containing a substantially pure polypeptide having3-hydroxypropionyl-CoA dehydratase activity can be used to catalyze theformation of 3-HP-CoA, a precursor to 3-HP. Further, cell-free extractscontaining a polypeptide having enzymatic activity can be used alone orin combination with purified polypeptides and/or cells to produce 3-HP.For example, a cell-free extract containing a polypeptide having CoAtransferase activity can be used to form lactyl-CoA, while amicroorganism containing polypeptides have the enzymatic activitiesnecessary to catalyze the reactions needed to form 3-HP from lactyl-CoAcan be used to produce 3-HP. Any method can be used to produce acell-free extract. For example, osmotic shock, sonication, and/or arepeated freeze-thaw cycle followed by filtration and/or centrifugationcan be used to produce a cell-free extract from intact cells.

It is noted that a cell, purified polypeptide, and/or cell-free extractcan be used to produce 3-HP that is, in turn, treated chemically toproduce another compound. For example, a microorganism can be used toproduce 3-HP, while a chemical process is used to modify 3-HP into aderivative such as polymerized 3-HP or an ester of 3-HP. Likewise, achemical process can be used to produce a particular compound that is,in turn, converted into 3-HP or other organic compound (e.g.,1,3-propanediol, acrylic acid, polymerized acrylate, esters of acrylate,esters of 3-HP, and polymerized 3-HP) using a cell, substantially purepolypeptide, and/or cell-free extract described herein. For example, achemical process can be used to produce acrylyl-CoA, while amicroorganism can be used convert acrylyl-CoA into 3-HP.

E. Fermentation of Cells to Produce Organic Acids

Typically, 3-HP is produced by providing a production cell, such as amicroorganism, and culturing the microorganism with culture medium suchthat 3-HP is produced. In general, the culture media and/or cultureconditions can be such that the microorganisms grow to an adequatedensity and produce 3-HP efficiently. For large-scale productionprocesses, any method can be used such as those described elsewhere(Manual of Industrial Microbiology and Biotechnology, 2^(nd) Edition,Editors: A. L. Demain and J. E. Davies, ASM Press; and Principles ofFermentation Technology, P. F. Stanbury and A. Whitaker, Pergamon).Briefly, a large tank (e.g., a 100 gallon, 200 gallon, 500 gallon, ormore tank) containing appropriate culture medium with, for example, aglucose carbon source is inoculated with a particular microorganism.After inoculation, the microorganisms are incubated to allow biomass tobe produced. Once a desired biomass is reached, the broth containing themicroorganisms can be transferred to a second tank. This second tank canbe any size. For example, the second tank can be larger, smaller, or thesame size as the first tank. Typically, the second tank is larger thanthe first such that additional culture medium can be added to the brothfrom the first tank. In addition, the culture medium within this secondtank can be the same as, or different from, that used in the first tank.For example, the first tank can contain medium with xylose, while thesecond tank contains medium with glucose.

Once transferred, the microorganisms can be incubated to allow for theproduction of 3-HP. Once produced, any method can be used to isolate the3-HP. For example, common separation techniques can be used to removethe biomass from the broth, and common isolation procedures (e.g.,extraction, distillation, and ion-exchange procedures) can be used toobtain the 3-HP from the microorganism-free broth. In addition, 3-HP canbe isolated while it is being produced, or it can be isolated from thebroth after the product production phase has been terminated.

F. Products Created from the Disclosed Biosynthetic Routes

The organic compounds produced from any of the steps provided in FIGS.1-5, 43-44, 54, and 55 can be chemically converted into other organiccompounds. For example, 3-HP can be hydrogenated to form 1,3propanediol, a valuable polyester monomer. Hydrogenating an organic acidsuch as 3-HP can be performed using any method such as those used tohydrogenate succinic acid and/or lactic acid. For example, 3-HP can behydrogenated using a metal catalyst. In another example, 3-HP can bedehydrated to form acrylic acid. Any method can be used to perform adehydration reaction. For example, 3-HP can be heated in the presence ofa catalyst (e.g., a metal or mineral acid catalyst) to form acrylicacid. Propanediol also can be created using polypeptides havingoxidoreductase activity (e.g., enzymes is the 1.1.1.- class of enzymes)in vitro or in vivo.

V. Overview of Methodology Used to Create Biosynthetic Pathways thatMake 3-HP from PEP

The invention provides methods of making 3-HP and related products fromPEP via the use of biosynthetic pathways. Illustrative examples includemethods involving the production of 3-HP via a lactate intermediate, amalonyl-CoA intermediate, and a 13-alanine intermediate.

A. Biosynthetic Pathway for Making 3-HP Through a Lactic AcidIntermediate

A biosynthetic pathway that allows for the production of 3-HP from PEPwas constructed (FIG. 1). This pathway involved using severalpolypeptides that were cloned and expressed as described herein. M.elsdenii cells (ATCC 17753) were used as a source of genomic DNA.Primers were used to identify and clone a nucleic acid sequence encodinga polypeptide having CoA transferase activity (SEQ ID NO: 1). Thepolypeptide was subsequently tested for enzymatic activity and found tohave CoA transferase activity.

Similarly, PCR primers were used to identify nucleic acid sequences fromM. elsdenii genomic DNA that encoded an E1 activator, E2 α, and E2 βpolypeptides (SEQ ID NOs: 9, 17, and 25, respectively). Thesepolypeptides were subsequently shown to have lactyl-CoA dehydrataseactivity.

Chloroflexus aurantiacus cells (ATCC 29365) were used as a source ofgenomic DNA. Initial cloning lead to the identification of nucleic acidsequences: OS17 (SEQ ID NO: 129) and OS19 (SEQ ID NO: 40). Subsequenceassays revealed that OS17 encodes a polypeptide having CoA synthaseactivity, dehydratase activity, and dehydrogenase activity(propionyl-CoA synthatase). Subsequence assays also revealed that OS19encodes a polypeptide having 3-hydroxypropionyl-CoA dehydratase activity(also referred to as acrylyl-CoA hydratase activity).

Several operons were constructed for use in E. coli. These operons allowfor the production of 3-HP in bacterial cells. Additional experimentsallowed for the expression of these polypeptide is yeast, which can beused to produce 3-HP.

B. Biosynthetic Pathway for Making 3-HP Through a Malonyl-CoAIntermediate

Another pathway leading to the production of 3-HP from PEP wasconstructed. This pathway used a polypeptide having acetyl CoAcarboxylase activity that was isolated from E. coli (Example 9), and apolypeptide having malonyl-CoA reductase activity that was isolated fromChloroflexus aurantacius (Example 10). The combination of these twopolypeptides allows for the production of 3-HP from acetyl-CoA (FIG.44).

Nucleic acid encoding a polypeptide having malonyl-CoA reductaseactivity (SEQ ID NO:140) was cloned, sequenced, and expressed. Thepolypeptide having malonyl-CoA reductase activity was then used to make3-HP.

C. Biosynthetic Pathways for Making 3-HP Through a β-alanineIntermediate

In general, prokaryotes and eukaryotes metabolize glucose via theEmbden-Meyerhof-Parnas pathway to PEP, a central metabolite in carbonmetabolism. The PEP generated from glucose is either carboxylated tooxaloacetate or is converted to pyruvate. Carboxylation of PEP tooxaloacetate can be catalyzed by a polypeptide having PEP carboxylaseactivity, a polypeptide having PEP carboxykinase activity, or apolypeptide having PEP transcarboxylase activity. Pyruvate that isgenerated from PEP by a polypeptide having pyruvate kinase activity canalso be converted to oxaloacetate by a polypeptide having pyruvatecarboxylase activity.

Oxaloacetate generated either from PEP or pyruvate can act as aprecursor for production of aspartic acid. This conversion can becarried out by a polypeptide having aspartate aminotransferase activity,which transfers an amino group from glutamate to oxaloacetate. Glutamateconsumed in this reaction can be regenerated by the action of apolypeptide having glutamate dehydrogenase activity or by the action ofa polypeptide having 4,4-aminobutyrate aminotransferase activity. Thedecarboxylation of aspartate to β-alanine is catalyzed by a polypeptidehaving aspartate decarboxylase activity. β-alanine produced through thisbiochemistry can be converted to 3-HP via two possible pathways. Thesepathways are provided in FIGS. 54 and 55.

The steps involved in the production of β-alanine can be the same forboth pathways. These steps can be accomplished by endogenouspolypeptides in the host cells which convert PEP to β-alanine, or thesesteps can be accomplished with recombinant DNA technology using knownpolypeptides such as polypeptides having PEP-carboxykinase activity(4.1.1.32), aspartate aminotransferase activity (2.6.1.1), and aspartatealpha-decarboxylase activity (4.1.1.11).

As depicted in FIG. 54, a polypeptide having CoA transferase activity(e.g., a polypeptide having a sequence set forth in SEQ ID NO:2) can beused to convert β-alanine to β-alanyl-CoA. β-alanyl-CoA can be convertedto acrylyl-CoA via a polypeptide having β-alanyl-CoA ammonia lyaseactivity (e.g., a polypeptide having a sequence set forth in SEQ IDNO:160). Acrylyl-CoA can be converted to 3-HP-CoA using a polypeptidehaving 3-HP-CoA dehydratase activity (e.g., a polypeptide having asequence set forth in SEQ ID NO:40). 3-HP-CoA can be converted into 3-HPvia a polypeptide having CoA transferase activity (e.g., a polypeptidehaving a sequence set forth in SEQ ID NO:2).

As depicted in FIG. 55, a polypeptide having 4,4-aminobutyrateaminotransferase activity (2.6.1.19) can be used to convert β-alanineinto malonate semialdehyde. The malonate semialdehyde can be convertedinto 3-HP using either a polypeptide having 3-hydroxypropionatedehydrogenase activity (1.1.1.159) or a polypeptide having3-hydroxyisobutyrate dehydrogenase activity.

EXAMPLES Example 1 Cloning Nucleic Acid Molecules that Encode aPolypeptide Having CoA Transferase Activity

Genomic DNA was isolated from Megasphaera elsdenii cells (ATCC 17753)grown in 1053 Reinforced Clostridium media under anaerobic conditions at37° C. in roll tubes for 12-14 hours. Once grown, the cells werepelleted, washed with 5 mL of a 10 mM Tris solution, and repelleted. Thepellet was resuspended in 1 mL of Gentra Cell Suspension Solution towhich 14.2 mg of lysozyme and 4 μL of 20 mg/mL proteinase K solution wasadded. The cell suspension was incubated at 37° C. for 30 minutes. Thegenomic DNA was than isolated using a Gentra Genomic DNA Isolation Kitfollowing the provided protocol. The precipitated genomic DNA wasspooled and air-dried for 10 minutes. The genomic DNA was suspended in500 μL of a 10 mM Tris solution and stored at 4° C.

Two degenerate forward (CoAF1 and CoAF2) and three degenerate reverse(CoAR1, CoAR2, and CoAR3) PCR primers were designed based on conservedacetoacetyl CoA transferase and propionate CoA transferase sequences(CoAF1 5′-GAAWSCGGYSCNATYGGYGG-3′, SEQ ID NO: 49; CoAF25′-TTYTGYGGYRSBTTYACBGCWGG-3′, SEQ ID NO: 50; CoAR15′-CCWGCVGTRAAVSYRCCRCARAA-3′, SEQ ID NO: 51; CoAR25′-AARACDSMRCGTTCVGTRATRTA-3′, SEQ ID NO: 52; and CoAR35′-TCRAYRCCSGGWGCRAYTTC-3′, SEQ ID NO: 53). The primers were used in alllogical combinations in PCR using Taq polymerase (Roche MolecularBiochemicals, Indianapolis, Ind.) and 1 ng of genomic DNA per μLreaction mix. PCR was conducted using a touchdown PCR program with 4cycles at an annealing temperature of 59° C., 4 cycles at 57° C., 4cycles at 55° C., and 18 cycles at 52° C. Each cycle used an initial30-second denaturing step at 94° C. and a 3 minute extension at 72° C.The program had an initial denaturing step for 2 minutes at 94° C. and afinal extension step of 4 minutes at 72° C. Time allowed for annealingwas 45 seconds. The amounts of PCR primer used in the reactions wereincreased 2-8 fold above typical PCR amounts depending on the amount ofdegeneracy in the 3′ end of the primer. In addition, separate PCRreactions containing each individual primer were made to identify PCRproducts resulting from single degenerate primers. Each PCR product (25μL) was separated by electrophoresis using a 1% TAE (Tris-acetate-EDTA)agarose gel.

The CoAF1-CoAR2, CoAF1-CoAR3, CoAF2-CoAR2, and CoAF2-CoAR3 combinationsproduced a band of 423, 474, 177, and 228 bp, respectively. These bandsmatched the sizes based on other CoA transferase sequences. No band wasvisible from the individual primer control reactions. The CoAF1-CoAR3fragment (474 bp) was isolated and purified using a Qiagen GelExtraction Kit (Qiagen Inc., Valencia, Calif.). Four μL of the purifiedband was ligated into pCRII vector and transformed into TOP10 E. colicells by heat-shock using a TOPO cloning procedure (Invitrogen,Carlsbad, Calif.). Transformations were plated on LB media containing100 μg/mL of ampicillin (Amp) and 50 μg/mL of5-Bromo-4-Chloro-3-Indolyl-B-D-Galactopyranoside (X-gal). Single, whitecolonies were plated onto fresh media and screened in a PCR reactionusing the CoAF1 and CoAR3 primers to confirm the presence of the insert.

Plasmid DNA obtained using a QiaPrep Spin Miniprep Kit (Qiagen, Inc) wasquantified and used for DNA sequencing with M13R and M13F primers.Sequence analysis revealed that the CoAF1-CoAR3 fragment shared sequencesimilarity with acetoacetyl CoA transferase sequences.

Genome walking was performed to obtain the complete coding sequence. Thefollowing primers for genome walking in both upstream and downstreamdirections were designed using the portion of the 474 bp CoAF1-CoAR3fragment sequence that was internal to the degenerate primers (COAGSP1F5′-GAATGTTTACTTCTGCGGCACCTTCAC-3′, SEQ ID NO:54; COAGSP2F5′-GACCAGATCACTTTCAACGGTTCCTATG-3′, SEQ ID NO:55; COAGSP1R5′-GCATAGGAACCGTTGAAAGTGATCTGG-3′, SEQ ID NO:56; and COAGSP2R5′-GTTAGTACCGAACTTGCTGACGTTGATG-3′, SEQ ID NO:57). The COAGSP1F andCOAGSP2F primers face downstream, while the COAGSP1R and COAGSP2Rprimers face upstream. In addition, the COAGSP2F and COAGSP2R primersare nested inside the COAGSP1F and COAGSP1R primers. Genome walking wasperformed using the Universal Genome Walking kit (ClonTech Laboratories,Inc., Palo Alto, Calif.) with the exception that additional librarieswere generated with enzymes Nru I, Sca I, and Hinc II. First round PCRwas conducted in a Perkin Elmer 2400 Thermocycler with 7 cycles of 2seconds at 94° C. and 3 minutes at 72° C., and 36 cycles of 2 seconds at94° C. and 3 minutes at 65° C. with a final extension at 65° C. for 4minutes. Second round PCR used 5 cycles of 2 seconds at 94° C. and 3minutes at 72° C., and 20 cycles of 2 seconds at 94° C. and 3 minutes at65° C. with a final extension at 65° C. for 4 minutes. The first andsecond round product (20 μL) was separated by electrophoresis on a 1%TAE agarose gel. Amplification products were obtained with the Stu Ilibrary for the reverse direction. The second round product of 1.5 Kbfrom this library was gel purified, cloned, and sequenced. Sequenceanalysis revealed that the sequence derived from genome walkingoverlapped with the CoAF1-CoAR3 fragment and shared sequence similaritywith other sequences such as acetoacetyl CoA transferase sequences(FIGS. 8-9).

Nucleic acid encoding the CoA transferase (propionyl-CoA transferase orpct) from Megasphaera elsdenii was PCR amplified from chromosomal DNAusing following PCR program: 25 cycles of 95° C. for 30 seconds todenature, 50° C. for 30 seconds to anneal, and 72° C. for 3 minutes forextension (plus 2 seconds per cycle). The primers used were designatedPCT-1.114 (5′-ATGAGAAAAGTAGAAATCATTAC-3′; SEQ ID NO:58) and PCT-2.2045(5′-GGCGGAAGTTGACGATAATG-3′; SEQ ID NO:59). The resulting PCR product(about 2 kb as judged by agarose gel electrophoresis) was purified usinga Qiagen PCR purification kit (Qiagen Inc., Valencia, Calif.). Thepurified product was ligated to pETBlue-1 using the Perfectly Bluntcloning Kit (Novagen, Madison, Wis.). The ligation reaction wastransformed into NovaBlue chemically competent cells (Novagen, Madison,Wis.) that were spread on LB agar plates supplemented with 50 μg/mLcarbenicillin, 40 μg/mL IPTG, and 40 μg/mL X-Gal. White colonies wereisolated and screened for the presence of inserts by restrictionmapping. Isolates with the correct restriction pattern were sequencedfrom each end using the primers pETBlueUP and pETBlueDOWN (Novagen) toconfirm the sequence at the ligation points.

The plasmid was transformed into Tuner (DE3) pLacI chemically competentcells (Novagen, Madison, Wis.), and expression from the constructtested. Briefly, a culture was grown overnight to saturation and diluted1:20 the following morning in fresh LB medium with the appropriateantibiotics. The culture was grown at 37° C. with aeration to an OD₆₀₀of about 0.6. The culture was induced with IPTG at a final concentrationof 100 μM. The culture was incubated for an additional two hours at 37°C. with aeration. Aliquots were taken pre-induction and 2 hourspost-induction for SDS-PAGE analysis. A band of the expected molecularweight (55,653 Daltons predicted from the sequence) was observed afterIPTG treatment. This band was not observed in cells containing a plasmidlacking the nucleic acid encoding the transferase.

Cell free extracts were prepared to assess enzymatic activity. Briefly,the cells were harvested by centrifugation and disrupted by sonication.The sonicated cell suspension was centrifuged to remove cell debris, andthe supernatant was used in the assays.

Transferase activity was measured in the following assay. The assaymixture used contained 100 mM potassium phosphate buffer (pH 7.0), 200mM sodium acetate, 1 mM dithiobisnitrobenzoate (DTNB), 500 μMoxaloacetate, 25 μM CoA-ester substrate, and 3 μg/mL citrate synthase.If present, the CoA transferase transfers the CoA from the CoA ester toacetate to form acetyl-CoA. The added citrate synthase condensesoxaloacetate and acetyl-CoA to form citrate and free CoASH. The freeCoASH complexes with DTNB, and the formation of this complex can bemeasured by a change in the optical density at 412 nm. The activity ofthe CoA transferase was measured using the following substrates:lactyl-CoA, propionyl-CoA, acrylyl-CoA, and 3-hydroxypropionyl-CoA. Theunits/mg of protein was calculated using the following formula:

(ΔE/min*V _(f)*dilution factor)/(V _(S)*14.2)=units/mL

where ΔE/min is the change in absorbance per minute at 412 nm, V_(f) isthe final volume of the reaction, and V_(S) is the volume of sampleadded. The total protein concentration of the cell free extract wasabout 1 mg/mL so the units/mL equals units/mg.

Cell free extracts from cells containing nucleic acid encoding the CoAtransferase exhibited CoA transferase activity (Table 2). The observedCoA transferase activity was detected for the lactyl-CoA, propionyl-CoA,acrylyl-CoA, and 3-hydroxypropionyl-CoA substrates (Table 2). Thehighest CoA transferase activity was detected for lactyl-CoA andpropionyl-CoA.

TABLE 2 Substrate Units/mg Lactyl-CoA 211 Propionyl-CoA 144 Acrylyl-CoA118 3-Hydroxypropionyl-CoA 110

The following assay was performed to test whether the CoA transferaseactivity can use the same CoA substrate donors as recipients.Specifically, CoA transferase activity was assessed using aMatrix-assisted Laser Desorption/Ionization Time of Flight MassSpectrometry (MALDI-TOF MS) Voyager RP workstation (PerSeptiveBiosystems). The following five reactions were analyzed:

1) acetate+lactyl-CoA→lactate+acetyl-CoA

2) acetate+propionyl-CoA→propionate+acetyl-CoA

3) lactate+acetyl-CoA→acetate+lactyl-CoA

4) lactate+acrylyl-CoA→acrylate+lactyl-CoA

5) 3-hydroxypropionate+lactyl-CoA→lactate+3-hydroxypropionyl-CoA

MALDI-TOF MS was used to measure simultaneously the appearance of theproduct CoA ester and the disappearance of the donor CoA ester. Theassay buffer contained 50 mM potassium phosphate (pH 7.0), 1 mM CoAester, and 100 mM respective acid salt. Protein from a cell free extractprepared as described above was added to a final concentration of 0.005mg/mL. A control reaction was prepared from a cell free extract preparedfrom cells lacking the construct containing the CoA transferase-encodingnucleic acid. For each reaction, the cell free extract was added last tostart the reaction. Reactions were allowed to proceed at roomtemperature and were stopped by adding 1 volume 10% trifluoroacetic acid(TFA). The reaction mixtures were purified prior to MALDI-TOF MSanalysis using Sep Pak Vac C₁₈ 50 mg columns (Waters, Inc.). The columnswere conditioned with 1 mL methanol and equilibrated with two washes of1 mL 0.1% TFA. Each sample was applied to the column, and the flowthrough was discarded. The column was washed twice with 1 mL 0.1% TFA.The sample was eluted in 200 μL 40% acetonitrile, 0.1% TFA. Theacetonitrile was removed by centrifugation in vacuo. Samples wereprepared for MALDI-TOF MS analysis by mixing 1:1 with 110 mM sinapinicacid in 0.1% TFA, 67% acetonitrile. The samples were allowed to air dry.

In reaction #1, the control sample exhibited a main peak at a molecularweight corresponding to lactyl-CoA (MW 841). There was a minor peak atthe molecular weight corresponding to acetyl-CoA (MW 811). This minorpeak was determined to be the left-over acetyl-CoA from the synthesis oflactyl-CoA. The reaction #1 sample containing the cell extract fromcells transfected with the CoA transferase-encoding plasmid exhibitedcomplete conversion of lactyl-CoA to acetyl-CoA. No peak was observedfor lactyl-CoA. This result indicates that the CoA transferase activitycan transfer CoA from lactyl-CoA to acetate to form acetyl-CoA.

In reaction #2, the control sample exhibited a dominant peak at amolecular weight corresponding to propionyl-CoA (MW 825). The reaction#2 sample containing the cell extract from cells transfected with theCoA transferase-encoding plasmid exhibited a dominant peak at amolecular weight corresponding to acetyl-CoA (MW 811). No peak wasobserved for propionyl-CoA. This result indicates that the CoAtransferase activity can transfer CoA from propionyl-CoA to acetate toform acetyl-CoA.

In reaction #3, the control sample exhibited a dominant peak at amolecular weight corresponding to acetyl-CoA (MW 811). The reaction #3sample containing the cell extract from cells transfected with the CoAtransferase-encoding plasmid exhibited a peak corresponding tolactyl-CoA (MW 841). The peak corresponding to acetyl-CoA did notdisappear. In fact, the ratio of the size of the two peaks was about1:1. The observed appearance of the peak corresponding to lactyl-CoAdemonstrates that the CoA transferase activity catalyzes reaction #3.

In reaction #4, the control sample exhibited a dominant peak at amolecular weight corresponding to acrylyl-CoA (MW 823). The reaction #4sample containing the cell extract from cells transfected with the CoAtransferase-encoding plasmid exhibited a dominant peak corresponding tolactyl-CoA (MW 841). This result demonstrates that the CoA transferaseactivity catalyzes reaction #4.

In reaction #5, deuterated lactyl-CoA was used to detect the transfer ofCoA from lactate to 3-hydroxypropionate since lactic acid and 3-HP havethe same molecular weight as do their respective CoA esters. Usingdeuterated lactyl-CoA allowed for the differentiation between lactyl-CoAand 3-hydroxypropionate using MALDI-TOF MS. The control sample exhibiteda diffuse group of peaks at molecular weights ranging from MW 841 to 845due to the varying amounts of hydrogen atoms that were replaced withdeuterium atoms. In addition, a significant peak was observed at amolecular weight corresponding to acetyl-CoA (MW 811). This peak wasdetermined to be the left-over acetyl-CoA from the synthesis oflactyl-CoA. The reaction #5 sample containing the cell extract fromcells transfected with the CoA transferase-encoding plasmid exhibited adominant peak at a molecular weight corresponding to3-hydroxypropionyl-CoA (MW 841) as opposed to a group of peaks rangingfrom MW 841 to 845. This result demonstrates that the CoA transferasecatalyzes reaction #5.

Example 2 Cloning Nucleic Acid Molecules that Encode a MultiplePolypeptide Complex Having Lactyl-CoA Dehydratase Activity

The following methods were used to clone an E1 activator polypeptide.Briefly, four degenerate forward and five degenerate reverse PCR primerswere designed based on conserved sequences of E1 activator proteinhomologs (E1F1 5′-GCWACBGGYTAYGGYCG-3′, SEQ ID NO:60; E1F25′-GTYRTYGAYRTYGGYGGYCAGGA-3′, SEQ ID NO:61; E1F35′-ATGAACGAYAARTGYGCWGCWGG-3′, SEQ ID NO:62; E1F45′-TGYGCWGCWGGYACBGGYCGYTT-3′, SEQ ID NO:63;E1R15′-TCCTGRCCRCCRAYRTCRAYRAC-3′, SEQ ID NO:64;E1R25′-CCWGCWGCRCAYTTRTCGTTCAT-3′, SEQ ID NO:65;E1R35′-AARCGRCCVGTRCCWGCWGCRCA-3′, SEQ ID NO:66;E1R45′-GCTTCGSWTTCRACRATGSW-3′, SEQ ID NO:67; andE1R55′-GSWRATRACTTCGCWTTCWGCRAA-3′, SEQ ID NO:68).

The primers were used in all logical combinations in PCR using Taqpolymerase (Roche Molecular Biochemicals, Indianapolis, Ind.) and 1 ngof genomic DNA per μL reaction mix. PCR was conducted using a touchdownPCR program with 4 cycles at an annealing temperature of 60° C., 4cycles at 58° C., 4 cycles at 56° C., and 18 cycles at 54° C. Each cycleused an initial 30-second denaturing step at 94° C. and a 3 minuteextension step at 72° C. The program had an initial denaturing step for2 minutes at 94° C. and a final extension step of 4 minutes at 72° C.Time allowed for annealing was 45 seconds. The amounts of PCR primerused in the reactions were increased 2-10 fold above typical PCR amountsdepending on the amount of degeneracy in the 3′ end of the primer. Inaddition, separate PCR reactions containing each individual primer weremade to identify PCR product resulting from single degenerate primers.Each PCR product (25 μL) was separated by electrophoresis using a 1% TAE(Tris-acetate-EDTA) agarose gel.

The E1F2-E1R4, E1F2-E1R5, E1F3-E1R4, E1F3-E1R5, and E1F4-E1R4R2combinations produced a band of 195, 207, 144, 156, and 144 bp,respectively. These bands matched the expected size based on E1activator sequences from other species. No band was visible withindividual primer control reactions. The E1F2-E1R5 fragment (207 bp) wasisolated and purified using Qiagen Gel Extraction procedure (QiagenInc., Valencia, Calif.). The purified band (4 μL) was ligated into apCRII vector that then was transformed into TOP10 E. coli cells byheat-shock using a TOPO cloning procedure (Invitrogen, Carlsbad,Calif.). Transformations were plated on LB media containing 100 μg/mL ofampicillin (Amp) and 50 μg/mL of5-Bromo-4-Chloro-3-Indolyl-B-D-Galactopyranoside (X-gal). Single, whitecolonies were plated onto fresh media and screened in a PCR reactionusing the E1F2 and E1R5 primers to confirm the presence of the insert.Plasmid DNA was obtained from multiple colonies using a QiaPrep SpinMiniprep Kit (Qiagen, Inc). Once obtained, the plasmid DNA wasquantified and used for DNA sequencing with M13R and M13F primers.Sequence analysis revealed a nucleic acid sequence encoding apolypeptide and revealed that the E1F2-E1R5 fragment shared sequencesimilarity with E1 activator sequences (FIGS. 12-13).

Genome walking was performed to obtain the complete coding sequence ofE2 α and β subunits. Briefly, four primers for performing genome walkingin both upstream and downstream directions were designed using theportion of the 207 bp E1F2-E1R5 fragment sequence that was internal tothe EIF2 and E1R5 degenerate primers (E1GSP1F5′-ACGTCATGTCGAAGGTACTGGAAATCC-3′, SEQ ID NO:69; E1GSP2F5′-GGGACTGGTACTTCAAATCGAAGCATC-3′, SEQ ID NO:70; E1GSP1R5′-TGACGGCAGCGGGATGCTTCGATTTGA-3′, SEQ ID NO:71; and E1GSP2R5′-TCAGACATGGGGATTTCCAGTACCTTC-3′, SEQ ID NO:72). The E1GSP1F andE1GSP2F primers face downstream, while the E1GSP1R and E1GSP2R primersface upstream. In addition, the E1GSP2F and E1GSP2R primers are nestedinside the E1GSP1F and E1GSP1R primers.

Genome walking was performed using the Universal Genome Walking Kit(ClonTech Laboratories, Inc., Palo Alto, Calif.) with the exception thatadditional libraries were generated with enzymes Nru I, Sca I, and HincII. First round PCR was performed in a Perkin Elmer 2400 Thermocyclerwith 7 cycles of 2 seconds at 94° C. and 3 minutes at 72° C., and 36cycles of 2 seconds at 94° C. and 3 minutes at 65° C. with a finalextension at 65° C. for 4 minutes. Second round PCR used 5 cycles of 2seconds at 94° C. and 3 minutes at 72° C., and 20 cycles of 2 seconds at94° C. and 3 minutes at 65° C. with a final extension at 65° C. for 4minutes. The first and second round product (20 μL) was separated byelectrophoresis using 1% TAE agarose gel. Amplification products wereobtained with the Stu I library for both forward and reverse directions.The second round product of about 1.5 kb for forward direction and 3 kbfragment for reverse direction from the Stu I library were gel purified,cloned, and sequenced. Sequence analysis revealed that the sequencederived from genome walking overlapped with the E1F2-E1R5 fragment.

To obtain additional sequence, a second genome walk was performed usinga first round primer (E1GSPF5 5′-CCGTGTTACTTGGGAAGGTATCGCTGTCTG-3′, SEQID NO:73) and a second round primer (E1GSPF65′-GCCAATGAAGGAGGAAA-CCACTAATGAGTC-3′, SEQ ID NO:74). The genome walkwas performed using the NruI, ScaI, and HincII libraries. In addition,ClonTech's Advantage-Genomic Polymerase was used for the PCR. Firstround PCR was performed in a Perkin Elmer 2400 Thermocycler with aninitial denaturing step at 94° C. for 2 minutes, 7 cycles of 2 secondsat 94° C. and 3 minutes at 72° C., and 36 cycles of 2 seconds at 94° C.and 3 minutes at 65° C. with a final extension at 65° C. for 4 minutes.Second round PCR used 5 cycles of 2 seconds at 94° C. and 3 minutes at72° C., and 20 cycles of 2 seconds at 94° C. and 3 minutes at 65° C.with a final extension at 65° C. for 4 minutes. The first and secondround product (20 μL) was separated by electrophoresis on a 1% agarosegel. An about 1.5 kb amplification product was obtained from secondround PCR of the HincII library. This band was gel purified, cloned, andsequenced. Sequence analysis revealed that it overlapped with thepreviously obtained genome walk fragment. In addition, sequence analysisrevealed a nucleic acid sequence encoding an E2 α subunit that sharessequence similarities with other sequences (FIGS. 16-17). Further,sequence analysis revealed a nucleic acid sequence encoding an E2 βsubunit that shares sequence similarities with other sequences (FIGS.20-21).

Additional PCR and sequence analysis revealed the order of polypeptideencoding sequences within the region containing the lactyl-CoAdehydratase-encoding sequences. Specifically, the E1GSP1F and COAGSP1Rprimer pair and the COAGSP1F and E1GSP1R primer pair were used toamplify fragments that encode both the CoA transferase and E1 activatorpolypeptides. Briefly, M. elsdenii genome DNA (1 ng) was used as atemplate. The PCR was conducted in Perkin Elmer 2400 Thermocycler usingLong Template Polymerase (Roche Molecular Biochemicals, Indianapolis,Ind.). The PCR program used was as follows: 94° C. for 2 minutes; 29cycles of 94° C. for 30 seconds, 61° C. for 45 seconds, and 72° C. for 6minutes; and a final extension of 72° C. for 10 minutes. Both PCRproducts (20 μL) were separated on a 1% agarose gel. An amplificationproduct (about 1.5 kb) was obtained using the COAGSP1F and E1GSP1Rprimer pair. This product was gel purified, cloned, and sequenced (FIG.22).

The organization of the M. elsdenii operon containing the lactyl-CoAdehydratase-encoding sequences was determined to containing thefollowing polypeptide-encoding sequences in the following order: CoAtransferase (FIG. 6), ORFX (FIG. 23), E1 activator protein of lactyl-CoAdehydratase (FIG. 10), E2 α subunit of lactyl-CoA dehydratase (FIG. 14),E2 β subunit of lactyl-CoA dehydratase (FIG. 18), and truncated CoAdehydrogenase (FIG. 25).

The lactyl-CoA dehydratase (lactyl-CoA dehydratase or lcd) from M.elsdenii was PCR amplified from chromosomal DNA using the followingprogram: 94° C. for 2 minutes; 7 cycles of 94° C. for 30 seconds, 47° C.for 45 seconds, and 72° C. for 3 minutes; 25 cycles of 94° C. for 30seconds, 54° C. for 45 seconds, and 72° C. for 3 minutes; and 72° C. for7 minutes. One primer pair was used (OSNBE1F5′-GGGAATTCCATATGAAAACTGTGTATACTCTC-3′, SEQ ID NO:75 and OSNBE1R5′-CGACGGATCCTTAGAGGATTTCCGAGAAAGC-3′, SEQ ID NO:76). The amplifiedproduct (about 3.2 kb) was separated on 1% agarose gel, cut from thegel, and purified with a Qiagen Gel Extraction kit (Qiagen, Valencia,Calif.). The purified product was digested with Nde I and BamHIrestriction enzymes and ligated into pET11a vector (Novagen) digestedwith the same enzymes. The ligation reaction was transformed intoNovaBlue chemically competent cells (Novagen) that then were spread onLB agar plates supplemented with 50 μg/mL carbenicillin. Isolatedindividual colonies were screened for the presence of inserts byrestriction mapping. Isolates with the correct restriction pattern weresequenced from each end using Novagen primers (T7 promoter primer#69348-3 and T7 terminator primer #69337-3) to confirm the sequence atthe ligation points.

A plasmid having the correct insert was transformed into Tuner (DE3)pLacI chemically competent cells (Novagen, Madison, Wis.). Expressionfrom this construct was tested as follows. A culture was grown overnightto saturation and diluted 1:20 the following morning in fresh LB mediumwith the appropriate antibiotics. The culture was grown at 37° C. withaeration to an OD₆₀₀ of about 0.6. The culture was induced with IPTG ata final concentration of 100 μM. The culture was incubated for anadditional two hours at 37° C. with aeration. Aliquots were takenpre-induction and 2 hours post-induction for SDS-PAGE analysis. Bands ofthe expected molecular weight (27,024 Daltons for the E1 subunit, 48,088Daltons for the E2 α subunit, and 42,517 Daltons for the E2 βsubunit—all predicted from the sequence) were observed. These bands werenot observed in cells containing a plasmid lacking the nucleic acidencoding the three components of the lactyl-CoA dehydratase.

Cell free extracts were prepared by growing cells in a sealed serumbottle overnight at 37° C. Following overnight growth, the cultures wereinduced with 1 mM IPTG (added using anaerobic technique) and incubatedan additional 2 hours at 37° C. The cells were harvested bycentrifugation and disrupted by sonication under strict anaerobicconditions. The sonicated cell suspension was centrifuged to remove celldebris, and the supernatant was used in the assays. The buffer used forcell resuspension/sonication was 50 mM Tris-HCl (pH 7.5), 200 μM ATP, 7mM Mg(SO₄), 4 mM DTT, 1 mM dithionite, and 100 μM NADH.

Dehydratase activity was detected with MALDI-TOF MS. The assay wasconducted in the same buffer as above with 1 mM lactyl-CoA or 1 mMacrylyl-CoA added and about 5 mg/mL cell free extract. Prior toMALDI-TOF MS analysis, samples were purified using Sep Pak Vac C₁₈columns (Waters, Inc.) as described in Example 1. The following tworeactions were analyzed:

1) acrylyl-CoA→lactyl-CoA

2) lactyl-CoA→acrylyl-CoA

In reaction #1, the control sample exhibited a peak at a molecularweight corresponding to acrylyl-CoA (MW 823). The reaction #1 samplecontaining the cell extract from cells transfected with thedehydratase-encoding plasmid exhibited a major peak at a molecularweight corresponding to lactyl-CoA (MW 841). This result indicates thatthe dehydratase activity can convert acrylyl-CoA into lactyl-CoA.

To detect dehydratase activity on lactyl-CoA, reaction #2 was carriedout in 80% D₂O. The control sample exhibited a peak at a molecularweight corresponding to lactyl-CoA (MW 841). The reaction #2 samplecontaining the cell extract from cells transfected with thedehydratase-encoding plasmid revealed a lactyl-CoA peak shifted to adeuterated form. This result indicates that the dehydratase enzyme isactive on lactyl-CoA. In addition, the results from both reactionsindicate that the dehydratase enzyme can catalyze thelactyl-CoA←→acrylyl-CoA reaction in both directions.

Example 3 Cloning Nucleic Acid Molecules that Encode a PolypeptideHaving 3-hydroxypropionyl CoA Dehydratase Activity

Genomic DNA was isolated from Chloroflexus aurantiacus cells (ATCC29365). Briefly, C. aurantiacus cells in 920 Chloroflexus medium weregrown in 50 mL cultures (Falcon 2070 polypropylene tubes) using anInnova 4230 Incubator, Shaker (New Brunswick Scientific; Edison, N.J.)at 50° C. with interior lights. Once grown, the cells were pelleted,washed with 5 mL of a 10 mM Tris solution, and re-pelleted. Genomic DNAwas isolated from the pelleted cells using a Gentra Genomic “Puregene”DNA isolation kit (Gentra Systems; Minneapolis, Minn.). Briefly, thepelleted cells were resuspended in 1 mL Gentra Cell Suspension Solutionto which 14.2 mg of lysozyme and 4 μL of 20 mg/mL proteinase K solutionwas added. The cell suspension was incubated at 37° C. for 30 minutes.The precipitated genomic DNA was recovered by centrifugation at 3500×gfor 25 minutes and air-dried for 10 minutes. The genomic DNA wassuspended in 300 μL of a 10 mM Tris solution and stored at 4° C.

The genomic DNA was used as a template in PCR amplification reactionswith primers designed based on conserved domains of crotonase homologsand a Chloroflexus aurantiacus codon usage table. Briefly, twodegenerate forward (CRF1 and CRF2) and three degenerate reverse (CRR1,CRR2, and CRR3) PCR primers were designed (CRF15′-AAYCGBCCVAARGCNCTSAAYGC-3′, SEQ ID NO:77; CRF2:5′-TTYGTBGCNGGYGCNGAYAT-3′, SEQ ID NO:78;CRR15′-ATRTCNGCRCCNGCVACRAA-3′, SEQ ID NO:79;CRR25′-CCRCCRCCSAGNGCRWARCCRTT-3′, SEQ ID NO:80; andCRR35′-SSWNGCRATVCGRATRTCRAC-3′, SEQ ID NO:81).

These primers were used in all logical combinations in PCR using Taqpolymerase (Roche Molecular Biochemicals; Indianapolis, Ind.) and 1 ngof the genomic DNA per μL reaction mix. The PCR was conducted using atouchdown PCR program with 4 cycles at an annealing temperature of 61°C., 4 cycles at 59° C., 4 cycles at 57° C., 4 cycles at 55° C., and 16cycles at 52° C. Each cycle used an initial 30-second denaturing step at94° C. and a 3-minute extension step at 72° C. The program also had aninitial denaturing step for 2 minutes at 94° C. and a final extensionstep of 4 minutes at 72° C. The time allowed for annealing was 45seconds. The amounts of PCR primer used in the reaction were increased4-12 fold above typical PCR amounts depending on the amount ofdegeneracy in the 3′ end of the primer. In addition, separate PCRreactions containing each individual primer were performed to identifyamplification products resulting from single degenerate primers. EachPCR product (25 μL) was separated by gel electrophoresis using a 1% TAE(Tris-acetate-EDTA) agarose gel.

The CRF1-CRR1 and CRF2-CRR2 combinations produced a unique band of about120 and about 150 bp, respectively. These bands matched the expectedsize based on crotonase genes from other species. No 120 bp or 150 bpband was observed from individual primer control reactions. Bothfragments (i.e., the 120 bp and 150 bp bands) were isolated and purifiedusing the Qiagen Gel Extraction kit (Qiagen Inc., Valencia, Calif.).Each purified fragment (4 μL) was ligated into pCRII vector that thenwas transformed into TOP10 E. coli cells by a heat-shock method using aTOPO cloning procedure (Invitrogen, Carlsbad, Calif.). Transformationswere plated on LB media containing 100 μg/mL of ampicillin (Amp) and 50μg/mL of 5-Bromo-4-Chloro-3-Indolyl-B-D-Galactopyranoside (X-gal).Single, white colonies were plated onto fresh media and screened in aPCR reaction using the CRF1 and CRR1 primers and the CRF2 and CRR2primers to confirm the presence of the desired insert. Plasmid DNA wasobtained from multiple colonies with the desired insert using a QiaPrepSpin Miniprep Kit (Qiagen, Inc.). Once obtained, the DNA was quantifiedand used for DNA sequencing with M13R and M13F primers. Sequenceanalysis revealed the presence of two different clones from the PCRproduct of about 150 bp. Each shared sequence similarity with crotonaseand hydratase sequences. The two clones were designated OS17 (157 bp PCRproduct) and OS19 (151 bp PCR product).

Genome walking was performed to obtain the complete coding sequence ofOS17. Briefly, primers for conducting genome walking in both upstreamand downstream directions were designed using the portion of the 157 bpCRF2-CRR2 fragment sequence that was internal to the CRF2 and CRR2degenerate primers (OS17F1 5′-CGCTG-ATATTCGCCAGTTGCTCGAAG-3′, SEQ IDNO:82; OS17F2 5′-CCCATCTTG-CTTTCCGCAAGATTGAGC-3′, SEQ ID NO:83; OS17F35′-CAATGGCCCTGCCGA-ATAACGCCCATCT-3′, SEQ ID NO:84;OS17R15′-CTTCGAGCAACTGGCGAA-TATCAGCG-3′, SEQ ID NO:85;OS17R25′-GCTCAATCTTGCGGAAAGCAAG-ATGGG-3′, SEQ ID NO:86; andOS17R35′-AGATGGGCGTTATTCGGCAGGGCC-ATTG-3′, SEQ ID NO:87). The OS17F1,OS17F3, and OS17F2 primers face downstream, while the OS17R2, OS17R3,and OS17R1 primers face upstream.

Genome walking was conducted using the Universal Genome Walking kit(ClonTech Laboratories, Inc., Palo Alto, Calif.) with the exception thatadditional libraries were generated with enzymes Nru I, Fsp I, and HincII. The first round PCR was conducted in a Perkin Elmer 2400Thermocycler with 7 cycles of 2 seconds at 94° C. and 3 minutes at 72°C., and 36 cycles of 2 seconds at 94° C. and 3 minutes at 66° C. with afinal extension at 66° C. for 4 minutes. Second round PCR used 5 cyclesof 2 seconds at 94° C. and 3 minutes at 72° C., and 20 cycles of 2seconds at 94° C. and 3 minutes at 66° C. with a final extension at 66°C. for 4 minutes. The first and second round amplification product (5μL) was separated by gel electrophoresis on a 1% TAE agarose gel. Afterthe second round PCR, an amplification product of about 0.4 kb wasobtained with the Fsp I library using the OS17R1 primer in the reversedirection, and an amplification product of about 0.6 kb was obtainedwith the Hinc II library using the OS17F2 primer in the forwarddirection. These PCR products were cloned and sequenced.

Sequence analysis revealed that the sequences derived from genomewalking overlapped with the CRF2-CRR2 fragment and shared sequencesimilarity with crotonase and hydratase sequences.

A second genome walking was performed to obtain additional sequences.Six primers were designed for this second genome walk (OS17F45′-AAGCTGGGTCTGATCGATGCCATTGCTACC-3′, SEQ ID NO:88; OS17F55′-CTCGATTATCG-CCCATCCACGTATCGAG-3′, SEQ ID NO:89; OS17F65′-TGGATGCAATCCG-CTATGGCATTATCCACG-3′, SEQ ID NO:90;OS17R45′-TCATTCAGTGCG-TTCACCGGCGGATTTGTC-3′, SEQ ID NO:91;OS17R55′-TCGATCCGGAAGT-AGCGATAGCGTTCGATG-3′, SEQ ID NO:92; and OS17R65′-CTTGGCTGCAAT-CTCTTCGAGCACTTCAGG-3′, SEQ ID NO:93). The OS17F4,OS17F5, and OS17F6 primers faced downstream, while the OS17R4, OS17R5,and OS17R6 primers faced upstream.

The second genome walk was performed using the same methods describedfor the first genome walk. After the second round of walking, anamplification product of about 2.3 kb was obtained with a Hinc IIlibrary using the OS 17R5 primer in the reverse direction, and anamplification product of about 0.6 kb was obtained with a Pvu II libraryusing the OS 17F5 primer in the forward direction. The PCR products werecloned and sequenced. Sequence analysis revealed that the sequencesderived from the second genome walking overlapped with the sequenceobtained during the first genome walking. In addition, the sequenceanalysis revealed a sequence with 3572 bp.

A BLAST search revealed that the polypeptide encoded by this sequenceshares sequence similarity with polypeptides having three differentactivities. Specifically, the beginning of the OS17 encoded-polypeptideshares sequence similarity with CoA-synthesases, the middle region ofthe OS17 encoded-polypeptide shares sequence similarity with enoyl-CoAhydratases, and the end region of the OS17 encoded-polypeptide sharessequence similarity with CoA-reductases.

A third genome walk was performed using four primers (OS17UP-65′-CATCAGAGGTAATCACCACTCGTGCA-3′, SEQ ID NO:94; OS17UP-75′-AAGTAGTAGGCCACCTCGTCGCCATA-3′, SEQ ID NO:95; OS17DN-15′-GCCAATCAGGCGCTGATCTATGTTCT-3′, SEQ ID NO:96; and OS17DN-25′-CTGATCTATGTTCTGGCCTCGGAGGT-3′, SEQ ID NO:97). The OS17UP-6 andOS17UP-7 primers face upstream, while the OS17DN-1 and OS17DN-2 primersface downstream. The third genome walk yielded an amplification productof about 1.2 kb with a Nru I library using the OS17UP-7 primer in thereverse direction. In addition, amplification products of about 4 kb andabout 1.1 kb were obtained with a Hinc II and Fsp I library,respectively, using the OS17DN-2 primer in the forward direction.Sequence analysis revealed a nucleic acid sequence encoding apolypeptide (FIGS. 27-28). The complete OS17 gene had 5466 nucleotidesand encoded a 1822 amino acid polypeptide. The calculated molecularweight of the OS17 polypeptide from the sequence was 201,346 (pI=5.71).

A BLAST search analysis revealed that the product of the OS17 nucleicacid has three different activities based on sequence similarity to (1)CoA-synthesases at the beginning of the OS17 sequence, (2) 3-HPdehydratases in the middle of the OS17 sequence, and (3) CoA-reductasesat the end of the OS17 sequence. Thus, the OS17 clone appeared to encodea single enzyme capable of catalyzing three distinct reactions leadingto the direct conversion of 3-hydroxypropionate to propionyl CoA:3-HP→3-HP-CoA+acrylyl-CoA→propionyl-CoA.

The OS17 gene from C. aurantiacus was PCR amplified from chromosomal DNAusing the following conditions: 94° C. for 3 minutes; 25 cycles of 94°C. for 30 seconds to denature, 54° C. for 30 seconds to anneal, and 68°C. for 6 minutes for extension; followed by 68° C. for 10 minutes forfinal extension. Two primers were used (OS17F5′-GGGAATTCCATATGATCGACACTGCG-3′, SEQ ID NO:136; and OS17R5′-CGAAGGATCCAACGATAATCGGCTCAGCAC-3′, SEQ ID NO:137). The resulting PCRproduct (˜5.6 Kb) was purified using Qiagen PCR purification kit (QiagenInc., Valencia, Calif.). The purified product was digested with NdeI andBamHI restriction enzymes, heated at 80° C. for 20 minutes to inactivatethe enzymes, purified using Qiagen PCR purification kit, and ligatedinto a pET11a vector (Novagen, Madison, Wis.) previously digested withNdeI and BamHI enzymes. The ligation reaction was transformed intoNovaBlue chemically competent cells (Novagen, Madison, Wis.) that werespread on LB agar plates supplemented with 50 μg/mL carbenicillin.Individual transformants were screened by PCR amplification of the OS17DNA with the OS17F and OS17R primers and conditions as described abovedirectly from colonies cells. Clones that yielded the 5.6 Kb productwere used for plasmid purification with Qiagen QiaPrep Spin Miniprep Kit(Qiagen, Inc). Resulting plasmids were transformed into E. coliBL21(DE3) cells, and OS17 polypeptide expression induced. The apparentmolecular weight of the OS17 polypeptide according to SDS gelelectrophoresis was about 190,000 Da.

To assay OS17 polypeptide function, a 100 mL culture ofBL21-DE3/pET11a-OS17 cells was started using 1 mL of overnight grownculture as an inoculum. The culture was grown to an OD of 0.5-0.6 andwas induced with 100 μM IPTG. After two and a half hours of induction,the cells were harvested by spinning at 8000 rpm in the floorcentrifuge. The cells were washed with 10 mM Tris-HCl (pH 7.8) andpassed twice through a French Press at a gauge pressure of 1000 psi. Thecell debris was removed by centrifugation at 15,000 rpm. The activity ofthe OS17 polypeptide was measured spectrophotometrically, and theproducts formed during this enzymatic transformation were detected byLC/MS. The assay mix was as follows (J. Bacteriol., 181:1088-1098(1999)):

Reagent Volume Final Conc. Tris-HCl (1000 mM, 7.8 pH) 10 μL 50 mM MgCl₂(100 mM) 10 μL 5 mM ATP (30 mM) 20 μL 3 mM KCl (100 mM) 20 μL 10 mMCoASH (5 mM) 20 μL 0.5 mM NAD(P)H 20 μL 0.5 mM 3-hydroxypropionate 2 μL1 mM Protein extract (7 mg/mL) 20 (40) μL 140 μg DI water 78 (58) μLTotal 200 μL

The initial rate of reaction was measured by monitoring thedisappearance of NAD(P)H at 340 nm. The activity of the OS17 polypeptidewas measured using 3-HP as the substrate. The units/mL of total proteinwas calculated using the formula set forth in Example 1. The activity ofthe expressed OS17 polypeptide was calculated to be 0.061 U/mL of totalprotein. The reaction products were purified using a Sep Pak Vac column(Waters). The column was conditioned with 1 mL methanol and washed twotimes with 0.5 mL 0.1% TFA. The sample was then applied to the column,and the column was washed two more times with 0.5 mL 0.1% TFA. Thesample was eluted with 200 μL of 40% acetonitrile, 0.1% TFA. Theacetonitrile was removed from the sample by vacuum centrifugation. Thereaction products were analyzed by LC/MS.

Analyses of thioesters namely propionyl CoA, acrylyl CoA, and 3 HP CoAfrom the above reaction were carried out using a Waters/Micromass ZQLC/MS instrument which had a Waters 2690 liquid chromatograph with aWaters 996 Photo-Diode Array (PDA) placed in series between thechromatograph and the single quadropole mass spectrometer. LCseparations were made using a 4.6×150 mm YMC ods-AQ (3 μm particles, 120Å pores) reversed-phase chromatography column at room temperature. CoAesters were eluted in Buffer A (25 mM ammonium acetate, 0.5% aceticacid) with a linear gradient of buffer B (acetonitrile, 0.5% aceticacid). A flow rate of 0.25 mL/minute was used, and photodiode array UVabsorbance was monitored from 200 to 400 nm. All parameters of theelectrospray MS system were optimized and selected based on generationof protonated molecular ions ([M+H]⁺) of the analytes of interest andproduction of characteristic fragment ions. The following instrumentalparameters were used for ESI-MS detection of CoA and organic acid-CoAthioesters in the positive ion mode; Extractor: 1 V; RF lens: 0 V;Source temperature: 100° C.; Desolvation temperature: 300° C.;Desolvation gas: 500 L/hour; Cone gas: 40 L/hour; Low mass resolution:13.0; High mass resolution: 14.5; Ion energy: 0.5; Multiplier: 650.Uncertainties for mass charge ratios (m/z) and molecular masses are±0.01%.

The enzyme assay mix from strains expressing the OS17 polypeptideexhibited peaks for propionyl CoA, acrylyl CoA, and 3-HP CoA with thepropionyl CoA peak being the dominant peak. These peaks where missing inthe enzyme assay mix obtained from the control strain, which carriedvector pET11a without an insert. These results indicate that the OS17polypeptide has CoA synthetase activity, CoA hydratase activity, anddehydrogenase activity.

Genome walking also was performed to obtain the complete coding sequenceof OS19. Briefly, primers for conducting genome walking in both upstreamand downstream directions were designed using the portion of the 151 bpCRF2-CRR2 fragment sequence that was internal to the CRF2 and CRR2degenerate primers (OS19F1 5′-GGCTGATATCAAAGCGATGGCCAATGC-3′, SEQ IDNO:98; OS19F2 5′-CCAC-GCCTATTGATATGCTCACCAGTG-3′, SEQ ID NO:99; OS19F35′-GCAAACCGG-TGATTGCTGCCGTGAATGG-3′, SEQ ID NO:100;OS19R15′-GCATTGGCCAT-CGCTTTGATATCAGCC-3′, SEQ ID NO:101;OS19R25′-CACTGGTGAGCATATC-AATAGGCGTGG-3′, SEQ ID NO:102; and OS19R35′-CCATTCACGGCAGCAA-TCACCGGTTTGC-3′, SEQ ID NO:103). The OS19F1, OS19F2,and OS19F3 primers face downstream, while the OS19R1, OS19R2, and OS19R3primers face upstream.

An amplification product of about 0.25 kb was obtained with the Fsp Ilibrary using the OS19R1 primer, while an amplification product of about0.65 kb was obtained with the Pvu II library using the OS19R1 primer. Inaddition, an amplification product of about 0.4 kb was obtained with thePvu II library using the OS19F3 primer. The PCR products were cloned andsequenced. Sequence analysis revealed that the sequences derived fromgenome walking overlapped with the CRF2-CRR2 fragment and sharedsequence similarity with crotonase and hydratase sequences. The obtainedsequences accounted for most of the coding sequence including the startcodon.

A second genome walk was performed to obtain additional sequence usingtwo primers (OS19F7 5′-TCATCATCGCCAGTGAAAACGCGCAGTTCG-3′, SEQ ID NO:104and OS19F8 5′-GGATCGCGCAAACCATTGCCACCAAATCAC-3′, SEQ ID NO:105). TheOS19F7 and OS19F8 primers face downstream.

An amplification product (about 0.7 kb) obtained from the Pvu II librarywas cloned and sequenced. Sequence analysis revealed that the sequencederived from the second genome walk overlapped with the sequenceobtained from the first genome walk and contained the stop codon. Thefull-length OS19 clone was found to share sequence similarity with othersequences such as crotonase and enoyl-CoA hydratase sequences (FIGS.32-33).

The OS19 clone was found to encode a polypeptide having3-hydroxypropionyl-CoA dehydratase activity also referred to asacrylyl-CoA hydratase activity. The nucleic acid encoding the OS19dehydratase from C. aurantiacus was PCR amplified from chromosomal DNAusing the following conditions: 94° C. for 3 minutes; 25 cycles of 94°C. for 30 seconds to denature, 56° C. for 30 seconds to anneal, and 68°C. for 1 minute for extension; and 68° C. for 5 minutes for finalextension. Two primers were used (OSACH35′-ATGAGTGAAGAGTCTCTGGTTCTCAGC-3′, SEQ ID NO:106 and OSACH25′-AGATCGCAATCGCTCGTGTATGTC-3′, SEQ ID NO:107).

The resulting PCR product (about 1.2 kb) was separated by agarose gelelectrophoresis and purified using Qiagen PCR purification kit (QiagenInc.; Valencia, Calif.). The purified product was ligated into pETBlue-1using the Perfectly Blunt cloning Kit (Novagen; Madison, Wis.). Theligation reaction was transformed into NovaBlue chemically competentcells (Novagen, Madison, Wis.) that then were spread on LB agar platessupplemented with 50 μg/mL carbenicillin, 40 μg/mL IPTG, and 40 μg/mLX-Gal. White colonies were isolated and screened for the presence ofinserts by restriction mapping. Isolates with the correct restrictionpattern were sequenced from each end using the primer pETBlueUP andpETBlueDOWN (Novagen) to confirm the sequence at the ligation points.

The plasmid containing the OS19 dehydratase-encoding sequence wastransformed into Tuner (DE3) pLacI chemically competent cells (Novagen,Madison, Wis.), and expression from the construct tested. Briefly, aculture was grown overnight to saturation and diluted 1:20 the followingmorning in fresh LB medium with the appropriate antibiotics. The culturewas grown at 37° C. and 250 rpm to an OD₆₀₀ of about 0.6. At this point,the culture was induced with IPTG at a final concentration of 1 mM. Theculture was incubated for an additional two hours at 37° C. and 250 rpm.Aliquots were taken pre-induction and 2 hours post-induction forSDS-PAGE analysis. A band of the expected molecular weight (27,336Daltons predicted from the sequence) was observed. This band was notobserved in cells containing a plasmid lacking the nucleic acid encodingthe hydratase.

Cell free extracts were prepared by growing cells as described above.The cells were harvested by centrifugation and disrupted by sonication.The sonicated cell suspension was centrifuged to remove cell debris, andthe supernatant was used in the assays. The ability of the3-hydroxypropionyl-CoA dehydratase to perform the following threereactions was measured using MALDI-TOF MS:

1) acrylyl-CoA→3-hydroxypropionyl-CoA

2) 3-hydroxypropionyl-CoA→acrylyl-CoA

3) crotonyl-CoA→3-hydroxybutyryl-CoA

The assay mixture contained 50 mM Tris-HCl (pH 7.5), 1 mM CoA ester, andabout 1 μg cell free extract. Reactions were allowed to proceed at roomtemperature and were stopped by adding 1 volume 10% trifluoroacetic acid(TFA). The reaction mixtures were purified prior to MALDI-TOF MSanalysis using Sep Pak Vac C₁₈ 50 mg columns (Waters, Inc.). The columnswere conditioned with 1 mL methanol and then equilibrated with twowashes of 1 mL 0.1% TFA. The sample was applied to the column, and theflow through was discarded. The column was washed twice with 1 mL 0.1%TFA. The sample was eluted in 200 μL 40% acetonitrile, 0.1% TFA. Theacetonitrile was removed by centrifugation in vacuo. Samples wereprepared for MALDI-TOF MS analysis by mixing 1:1 with 110 mM sinapinicacid in 0.1% TFA, 67% acetonitrile. The samples were allowed to air dry.

The conversion of acrylyl-CoA into 3-hydroxypropionyl-CoA catalyzed bythe 3-hydroxypropionyl-CoA dehydratase was detected using the MALDI-TOFMS technique. In reaction #1, the control sample exhibited a dominantpeak at a molecular weight corresponding to acrylyl-CoA (MW 823). Thereaction #1 sample containing the cell extract from cells transfectedwith the 3-hydroxypropionyl-CoA dehydratase-encoding plasmid exhibited adominant peak corresponding to 3-hydroxypropionyl-CoA (MW 841). Thisresult demonstrates that the 3-hydroxypropionyl-CoA dehydratase activitycatalyzes reaction #1.

To detect the conversion of 3-hydroxypropionyl-CoA into acrylyl-CoA,reaction #2 was carried out in 80% D₂O. The reaction #2 samplecontaining the cell extract from cells transfected with the3-hydroxypropionyl-CoA dehydratase-encoding plasmid revealedincorporation of deuterium in the 3-hydroxypropionyl-CoA molecule. Thisresult indicates that the 3-hydroxypropionyl-CoA dehydratase enzymecatalyzes reaction #2. In addition, the results from both #1 and #2reactions indicate that the 3-hydroxypropionyl-CoA dehydratase enzymecan catalyze the 3-hydroxypropinyl-CoA←→acrylyl-CoA reaction in bothdirections. It is noted that for both the #1 and #2 reactions, a peakwas observed at MW 811, due to leftover acetyl-CoA from the synthesis of3-hydroxypropionyl-CoA from 3-hydroxypropionate and acetyl-CoA.

The assays assessing conversion of crotonyl-CoA into3-hydroxybutyryl-CoA also were carried out in 80% D₂O. In reaction #3,the control sample exhibited a dominant peak at a molecular weightcorresponding to crotonyl-CoA (MW 837). This result indicated that thecrotonyl-CoA was not converted into other products. The reaction #3sample containing the cell extract from cells transfected with the3-hydroxypropionyl-CoA dehydratase-encoding plasmid exhibited a diffusegroup of peaks corresponding to deuterated 3-hydroxybutyryl-CoA (MW 855to MW 857). This result demonstrates that the 3-hydroxypropionyl-CoAdehydratase activity catalyzes reaction #3.

A series of control reactions were performed to confirm the specificityof the 3-hydroxypropionyl-CoA dehydratase. Lactyl-CoA (1 mM) was addedto the reaction mixture containing 100 mM Tris (pH 7.0) both in thepresence and the absence of the 3-hydroxypropionyl-CoA dehydratase. Inboth cases, the dominant peak observed had a molecular weightcorresponding to lactyl-CoA (MW 841). This result indicates thatlactyl-CoA is not affected by the presence of 3-hydroxypropionyl-CoAdehydratase activity even in the presence of D₂O meaning that the3-hydroxypropionyl-CoA dehydratase enzyme does not attach a hydroxylgroup at the alpha carbon position. The presence of3-hydroxypropionyl-CoA in an 80% D₂O reaction mixture resulted in ashift upon addition of the 3-hydroxypropionyl-CoA dehydratase activity.In the absence of 3-hydroxypropionyl-CoA dehydratase activity, a peakcorresponding to 3-hydroxypropionyl-CoA was observed in addition to apeak of MW 811. The MW 811 peak was due to leftover acetyl-CoA from thesynthesis of 3-hydroxypropionyl-CoA. In the presence of3-hydroxypropionyl-CoA dehydratase activity, a peak corresponding todeuterated 3-hydroxypropionyl-CoA was observed (MW 842) due to exchangeof a hydroxyl group during the conversion of 3-hydroxypropionyl-CoA toacrylyl-CoA and visa-versa. These control reactions demonstrate that the3-hydroxypropionyl-CoA dehydratase enzyme is active on3-hydroxypropionyl-CoA and not active on lactyl-CoA. In addition, theseresults demonstrate that the product of the acrylyl-CoA reaction is3-hydroxypropionyl-CoA not lactyl-CoA.

Example 4 Construction of Operon #1

The following operon was constructed and can be used to produce 3-HP inE. coli (FIG. 34). Briefly, the operon was cloned into a pET-11aexpression vector under the control of a T7 promoter (Novagen, Madison,Wis.). The pET-11a expression vector is a 5677 bp plasmid that uses theATG sequence of an NdeI restriction site as a start codon for inserteddownstream sequences.

Nucleic acid molecules encoding a CoA transferase and a lactyl-CoAdehydratase were amplified from Megasphaera elsdenii genomic DNA by PCR.Two primers were used to amplify the CoA transferase-encoding sequence(OSNBpctF 5′-GGGAATTCC-ATATGAGAAAAGTAGAAATCATTACAGCTG-3′, SEQ ID NO:108and OSCTE-2 5′-GAGAGTATACACAGTTTTCACCTCCTTTACAGCAGAGAT-3′, SEQ IDNO:109), and two primers were used to amplify the lactyl-CoAdehydratase-encoding sequence (OSCTE-15′-ATCTCTGCTGTAAAGGAGGTGAAAACTGTGTATACT-CTC-3′, SEQ ID NO:110 andOSEBH-2 5′-ACGTTGATCTCCTTGTACATTAGAGGATTTCCGAGAAAGC-3′, SEQ ID NO:111).A nucleic acid molecule encoding a 3-hydroxypropionyl-CoA dehydratasewas amplified from Chloroflexus aurantiacus genomic DNA of by PCR usingtwo primers (OSEBH-1 5′-GCTTTCTCGG-AAATCCTCTAATGTACAAGGAGATCAACGT-3′,SEQ ID NO:112 and OSHBR 5′-CGACGGATCCTCAACGACCACTGAAGTTGG-3′, SEQ IDNO:113).

PCR was conducted in a Perkin Elmer 2400 Thermocycler using 100 ng ofgenomic DNA and a mix of rTth polymerase (Applied Biosystems; FosterCity, Calif.) and Pfu Turbo polymerase (Stratagene; La Jolla, Calif.) in8:1 ratio. The polymerase mix ensured higher fidelity of the PCRreaction. The following PCR conditions were used: initial denaturationstep of 94° C. for 2 minutes; 20 cycles of 94° C. for 30 seconds, 54° C.for 30 seconds, and 68° C. for 2 minutes; and a final extension at 68°C. for 5 minutes. The obtained PCR products were gel purified using aQiagen Gel Extraction Kit (Qiagen, Inc.; Valencia, Calif.).

The CoA transferase, lactyl-CoA dehydratase (E1, E2 α subunit, and E2 βsubunit), and 3-hydroxypropionyl-CoA dehydratase PCR products wereassembled using PCR. The OSCTE-1 and OSCTE-2 primers as well as theOSEBH-1 and OSEBH-2 primers were complementary to each other. Thus, thecomplementary DNA ends could anneal to each other during the PCRreaction extending the DNA in both direction. To ensure the efficiencyof the assembly, two end primers (OSNBpctF and OSHBR) were added to theassembly PCR mixture, which contained 100 ng of each PCR product (i.e.,the PCR products from the CoA-transferase, lactyl-CoA dehydratase, and3-hydroxypropionyl-CoA dehydratase reactions) as well as the rTthpolymerase/Pfu Turbo polymerase mix described above. The following PCRconditions were used to assemble the products: 94° C. for 1 minute; 25cycles of 94° C. for 30 seconds, 55° C. for 30 seconds, and 68° C. for 6minutes; and a final extension at 68° C. for 7 minutes. The assembledPCR product was gel purified and digested with restriction enzymes (NdeIand BamHI). The sites for these restriction enzymes were introduced intothe assembled PCR product using the OSNBpctF (NdeI) and OSHBR (BamHI)primers. The digested PCR product was heated at 80° C. for 30 minutes toinactive the restriction enzymes and used directly for ligation intopET-11a vector.

The pET-11a vector was digested with NdeI and BamHI restriction enzymes,gel purified using a Qiagen Gel Extraction kit, treated with shrimpalkaline phosphatase (Roche Molecular Biochemicals; Indianapolis, Ind.)and used in a ligation reaction with the assembled PCR product. Theligation was performed at 16° C. overnight using T4 ligase (RocheMolecular Biochemicals; Indianapolis, Ind.). The resulting ligationreaction was transformed into NovaBlue chemically competent cells(Novagen; Madison, Wis.) using a heat-shock method. Once heat shocked,the cells were plated on LB plates supplemented with 50 μg/mLcarbenicillin. The plasmid DNA was purified from individual coloniesusing a QiaPrep Spin Miniprep Kit (Qiagen Inc., Valencia, Calif.) andanalyzed by digestion with NdeI and BamHI restriction enzymes.

Example 5 Construction of Operon #2

The following operon was constructed and can be used to produce 3-HP inE. coli (FIGS. 35A and B). Nucleic acid molecules encoding a CoAtransferase and a lactyl-CoA dehydratase were amplified from Megasphaeraelsdenii genomic DNA by PCR. Two primers were used to amplify the CoAtransferase-encoding sequence (OSNBpctF and OSCTE-2), and two primerswere used to amplify the lactyl-CoA dehydratase-encoding sequence(OSCTE-1 and OSNBe1R 5′-CGACGGATCCTTAGAGGATTT-CCGAGAAAGC-3′, SEQ IDNO:114). A nucleic acid molecule encoding a 3-hydroxypropionyl-CoAdehydratase was amplified from Chloroflexus aurantiacus genomic DNA ofby PCR using two primers (OSXNHF5′-GGTGTCT-AGAGACAGTCCTGTCGTTTATGTAGAAGGAG-3′, SEQ ID NO:115 and OSXNhR5′-GGGAATTCCATATGCGTAACTTCCTCCTGCTATCAACGACCACTGAAGTTGG-3′, SEQ IDNO:116).

PCR was conducted in a Perkin Elmer 2400 Thermocycler using 100 ng ofgenomic DNA and a mix of rTth polymerase (Applied Biosystems; FosterCity, Calif.) and Pfu Turbo polymerase (Stratagene; La Jolla, Calif.) in8:1 ratio. The polymerase mix ensured higher fidelity of the PCRreaction. The following PCR conditions were used: initial denaturationstep of 94° C. for 2 minutes; 20 cycles of 94° C. for 30 seconds, 54° C.for 30 seconds, and 68° C. for 2 minutes; and a final extension at 68°C. for 5 minutes. The obtained PCR products were gel purified using aQiagen Gel Extraction Kit (Qiagen, Inc.; Valencia, Calif.).

The CoA transferase and lactyl-CoA dehydratase (E1, E2 α subunit, and E2β subunit) PCR products were assembled using PCR. The OSCTE-1 andOSCTE-2 primers were complementary to each other. Thus, the 22nucleotides at the end of the CoA transferase sequence and the 22nucleotides at the beginning of the lactyl-CoA dehydratase could annealto each other during the PCR reaction extending the DNA in bothdirection. To ensure the efficiency of the assembly, two end primers(OSNBpctF and OSNBelR) were added to the assembly PCR mixture, whichcontained 100 ng of the CoA transferase PCR product, 100 ng oflactyl-CoA dehydratase PCR product, and the rTth polymerase/Pfu Turbopolymerase mix described above. The following PCR conditions were usedto assemble the products: 94° C. for 1 minute; 20 cycles of 94° C. for30 seconds, 54° C. for 30 seconds, and 68° C. for 5 minutes; and a finalextension at 68° C. for 6 minutes.

The assembled PCR product was gel purified and digested with restrictionenzymes (NdeI and BamHI). The sites for these restriction enzymes wereintroduced into the assembled PCR product using the OSNBpctF (NdeI) andOSNBelR (BamHI) primers. The digested PCR product was heated at 80° C.for 30 minutes to inactive the restriction enzymes and used directly forligation into a pET-11a vector.

The pET-11a vector was digested with NdeI and BamHI restriction enzymes,gel purified using a Qiagen Gel Extraction kit, treated with shrimpalkaline phosphatase (Roche Molecular Biochemicals; Indianapolis, Ind.)and used in a ligation reaction with the assembled PCR product. Theligation was performed at 16° C. overnight using T4 ligase (RocheMolecular Biochemicals; Indianapolis, Ind.). The resulting ligationreaction was transformed into NovaBlue chemically competent cells(Novagen; Madison, Wis.) using a heat-shock method. Once heat shocked,the cells were plated on LB plates supplemented with 50 μg/mLcarbenicillin. The plasmid DNA was purified from individual coloniesusing a QiaPrep Spin Miniprep Kit (Qiagen Inc., Valencia, Calif.) andanalyzed by digestion with NdeI and BamHI restriction enzymes. Thedigest revealed that the DNA fragment containing CoAtransferase-encoding and lactyl-CoA dehydratase-encoding sequences wascloned into the pET-11a vector.

The plasmid carrying the CoA transferase-encoding and lactyl-CoAdehydratase-encoding sequences (pTD) was digested with XbaI and NdeIrestriction enzymes, gel purified, and used for cloning the3-hydroxypropionyl-CoA dehydratase-encoding product upstream of the CoAtransferase-encoding sequence. Since this XbaI and NdeI digesteliminated a ribosome-binding site (RBS) from the pET-11a vector, a newhomologous RBS was cloned into the plasmid together with the3-hydroxypropionyl-CoA dehydratase-encoding product. Briefly, the3-hydroxypropionyl-CoA dehydratase-encoding PCR product was digestedwith XbaI and NdeI restriction enzymes, heated at 65° C. for 30 minutesto inactivate the restriction enzymes, and ligated into pTD. Theligation mixture was transformed into chemically competent NovaBluecells (Novagen) that then were plated on LB plates supplemented with 50μg/mL carbenicillin.

Individual colonies were selected, and the plasmid DNA obtained using aQiagen Spin Miniprep Kit. The obtained plasmids were digested with XbaIand NdeI restriction enzymes and analyzed by gel electrophoresis. pTDplasmids containing the inserted 3-hydroxypropionyl-CoAdehydratase-encoding PCR product were named pHTD. While expression ofthe lactyl-CoA hydratase, CoA transferase, and 3-hydroxypropionyl-CoAdehydratase sequences from pHTD was directed by a single T7 promoter,each coding sequence had an individual RBS upstream of their startcodon.

To ensure the correct assembly and cloning of the lactyl-CoA hydratase,CoA transferase, and 3-hydroxypropionyl-CoA dehydratase sequences intoone operon, both ends of the operon and all junctions between the codingsequences were sequenced. This DNA analysis revealed that the operon wasassembled correctly.

The pHTD plasmid was transformed into BL21(DE3) cells to study theexpression of the encoded sequences.

Example 6 Construction of Operons #3 and #4

Operon #3 (FIGS. 36A and B) and operon #4 (FIGS. 37A and B) eachposition the E1 activator at the end of the operon. Operon #3 contains aRBS between the 3-hydroxypropionyl-CoA dehydratase-encoding sequence andthe E1 activator-encoding sequence. In operon #4, however, the stopcodon of the 3-hydroxypropionyl-CoA dehydratase-encoding sequence isfused with the start codon of the E1 activator-encoding sequence asfollows: TAGTG. The absence of the RBS in operon #4 can decrease thelevel of E1 activator expression.

To construct operon #3, nucleic acid molecules encoding a CoAtransferase and a lactyl-CoA dehydratase were amplified from Megasphaeraelsdenii genomic DNA by PCR. Two primers were used to amplify the CoAtransferase-encoding sequence (OSNBpctF and OSHTR5′-ACGTTGATCTCCTTCTACATTATTTTTTCAGTCCCATG-3′, SEQ ID NO:117), twoprimers were used to amplify the E2 α and β subunits of the lactyl-CoAdehydratase-encoding sequence (OSEIIXNF5′-GGTGTCTAGAGTCAAAGGAGAGAACAAAATCATGAGTG-3′, SEQ ID NO:118 and OSEIIXNR5′-GGGAATTCCATATGCGTAACTTCCTCCTGCTATTAGAGGATTTCCGAGAAAGC-3′, SEQ IDNO:119), and two primers were used to amplify the E1 activator of thelactyl-CoA dehydratase-encoding sequence (OSHrEIF5′-TCAGTGGTCGTTGATCACGCTATAAAGAAAGGTGAAAACTGTGTATACTCTC-3′, SEQ IDNO:120 and OSEIBR 5′-CGACGGATCCCTTCCTTGGAGCTCATGCTTTC-3′, SEQ IDNO:121). A nucleic acid molecule encoding a 3-hydroxypropionyl-CoAdehydratase was amplified from Chloroflexus aurantiacus genomic DNA ofby PCR using two primers (OSTHF5′-CATGGGACTGAAAAAATAATGTAGAAGGAGATCAACGT-3′, SEQ ID NO:122 and OSEIrHR5′-GAGAGTATACACAGTTTTCACCTTTCTTTATAGCGTGATCAACGACCACTGA-3′, SEQ IDNO:123).

PCR was conducted in a Perkin Elmer 2400 Thermocycler using 100 ng ofgenomic DNA and a mix of rTth polymerase (Applied Biosystems; FosterCity, Calif.) and Pfu Turbo polymerase (Stratagene; La Jolla, Calif.) in8:1 ratio. The polymerase mix ensured higher fidelity of the PCRreaction. The following PCR conditions were used: initial denaturationstep of 94° C. for 2 minutes; 20 cycles of 94° C. for 30 seconds, 54° C.for 30 seconds, and 68° C. for 2 minutes; and a final extension at 68°C. for 5 minutes. The obtained PCR products were gel purified using aQiagen Gel Extraction Kit (Qiagen, Inc.; Valencia, Calif.).

The 3-hydroxypropionyl-CoA dehydratase and E1 activator PCR productswere assembled using PCR. The OSHrE1F and OSEIrHR primers werecomplementary to each other. Thus, the primers could anneal to eachother during the PCR reaction extending the DNA in both direction. Toensure the efficiency of the assembly, two end primers (OSTHF andOSE1BR) were added to the assembly PCR mixture, which contained 100 ngof the 3-hydroxypropionyl-CoA dehydratase PCR product, 100 ng of E1activator PCR product, and the rTth polymerase/Pfu Turbo polymerase mixdescribed above. The following PCR conditions were used to assemble theproducts: 94° C. for 1 minute; 20 cycles of 94° C. for 30 seconds, 54°C. for 30 seconds, and 68° C. for 1.5 minutes; and a final extension at68° C. for 5 minutes.

The assembled PCR product was gel purified and used in a second assemblyPCR with gel purified the CoA transferase PCR product. The OSTHF andOSHTR primers were complementary to each other. Thus, the complementaryDNA ends could anneal to each other during the PCR reaction extendingthe DNA in both direction. To ensure the efficiency of the assembly, twoend primers (OSNBpctF and OSEIBR) were added to the second assembly PCRmixture, which contained 100 ng of the purified 3-hydroxypropionyl-CoAdehydratase/EI PCR assembly, 100 ng of the purified CoA transferase PCRproduct, and the polymerase mix described above. The following PCRconditions were used to assemble the products: 94° C. for 1 minute; 20cycles of 94° C. for 30 seconds, 54° C. for 30 seconds, and 68° C. for 3minutes; and a final extension at 68° C. for 5 minutes.

The assembled PCR product was gel purified and digested with NdeI andBamHI restriction enzymes. The sites for these restriction enzymes wereintroduced into the assembled PCR products with the OSNBpctF (NdeI) andOSEIBR (BamHI) primers. The digested PCR product was heated at 80° C.for 30 minutes to inactive the restriction enzymes and used directly forligation into a pET11a vector.

The pET-11a vector was digested with NdeI and BamHI restriction enzymes,gel purified using a Qiagen Gel Extraction kit, treated with shrimpalkaline phosphatase (Roche Molecular Biochemicals; Indianapolis, Ind.)and used in a ligation reaction with the assembled PCR product. Theligation was performed at 16° C. overnight using T4 ligase (RocheMolecular Biochemicals; Indianapolis, Ind.). The resulting ligationreaction was transformed into NovaBlue chemically competent cells(Novagen; Madison, Wis.) using a heat-shock method. Once heat shocked,the cells were plated on LB plates supplemented with 50 μg/mLcarbenicillin. The plasmid DNA was purified from individual coloniesusing a QiaPrep Spin Miniprep Kit (Qiagen Inc.; Valencia, Calif.). Theresulting plasmids carrying the CoA transferase, 3-hydroxypropionyl-CoAdehydratase, and E1 activator sequences (pTHrEI) were digested with XbaIand NdeI, purified using gel electrophoresis and a Qiagen Gel Extractionkit, and used as a vector for cloning of the E2 α subunit/E2 β subunitPCR product.

The E2 α subunit/E2 β subunit PCR product was digested with the sameenzymes and ligated into the pTHrEI vector. The ligation reaction wasperformed at 16° C. overnight using T4 ligase (Roche MolecularBiochemicals; Indianapolis, Ind.). The ligation mixture was transformedinto chemically competent NovaBlue cells (Novagen) that then were platedon LB plates supplemented with 50 μg/mL carbenicillin. The plasmid DNAwas purified from individual colonies using a QiaPrep Spin Miniprep Kit(Qiagen Inc., Valencia, Calif.) and digested with XbaI and NdeIrestriction enzymes for gel electrophoresis analysis. The resultingplasmids carrying the constructed operon #3 (pEIITHrEI) were transformedinto BL21 (DE3) cells to study the expression of the cloned sequences.Electrospray mass spectrometry assay confirmed that extracts from thesecells have CoA transferase activity and 3-hydroxypropionyl-CoAdehydratase activity. Similar assays are used to confirm that extractsfrom these cells also have lactyl-CoA dehydratase activity.

To construct operon #4, nucleic acid molecules encoding a CoAtransferase and a lactyl-CoA dehydratase were amplified from Megasphaeraelsdenii genomic DNA by PCR. Two primers were used to amplify the CoAtransferase-encoding sequence (OSNBpctF and OSHTR), two primers wereused to amplify the E2 α and β subunits of the lactyl-CoAdehydratase-encoding sequence (OSEIIXNF and OSEIIXNR), and two primerswere used to amplify the E1 activator of the lactyl-CoAdehydratase-encoding sequence (OSHEIF5′-CCAACTTCAGTGGTCGTTAGTGAAAACTGTGTATACTCTC-3′, SEQ ID NO:124 andOSEIBR). A nucleic acid molecule encoding a 3-hydroxypropionyl-CoAdehydratase was amplified from Chloroflexus aurantiacus genomic DNA ofby PCR using two primers (OSTHF and OSEIHR5′-GAGAGTATACACAGTTTTCACTAACGACCACTGAAGTTGG-3′, SEQ ID NO:125).

PCR was conducted in a Perkin Elmer 2400 Thermocycler using 100 ng ofgenomic DNA and a mix of rTth polymerase (Applied Biosystems; FosterCity, Calif.) and Pfu Turbo polymerase (Stratagene; La Jolla, Calif.) in8:1 ratio. The polymerase mix ensured higher fidelity of the PCRreaction. The following PCR conditions were used: initial denaturationstep of 94° C. for 2 minutes; 20 cycles of 94° C. for 30 seconds, 54° C.for 30 seconds, and 68° C. for 2 minutes; and a final extension at 68°C. for 5 minutes. The obtained PCR products were gel purified using aQiagen Gel Extraction Kit (Qiagen, Inc.; Valencia, Calif.).

The 3-hydroxypropionyl-CoA dehydratase and E1 activator PCR productswere assembled using PCR. The OSHEIF and OSEIHR primers werecomplementary to each other. Thus, the primers could anneal to eachother during the PCR reaction extending the DNA in both direction. Toensure the efficiency of the assembly, two end primers (OSTHF andOSE1BR) were added to the assembly PCR mixture, which contained 100 ngof the 3-hydroxypropionyl-CoA dehydratase PCR product, 100 ng of E1activator PCR product, and the rTth polymerase/Pfu Turbo polymerase mixdescribed above. The following PCR conditions were used to assemble theproducts: 94° C. for 1 minute; 20 cycles of 94° C. for 30 seconds, 54°C. for 30 seconds, and 68° C. for 1.5 minutes; and a final extension at68° C. for 5 minutes.

The assembled PCR product was gel purified and used in a second assemblyPCR with gel purified the CoA transferase PCR product. The OSTHF andOSHTR primers were complementary to each other. Thus, the complementaryDNA ends could anneal to each other during the PCR reaction extendingthe DNA in both direction. To ensure the efficiency of the assembly, twoend primers (OSNBpctF and OSEIBR) were added to the second assembly PCRmixture, which contained 100 ng of the purified 3-hydroxypropionyl-CoAdehydratase/EI PCR assembly, 100 ng of the purified CoA transferase PCRproduct, and the polymerase mix described above. The following PCRconditions were used to assemble the products: 94° C. for 1 minute; 20cycles of 94° C. for 30 seconds, 54° C. for 30 seconds, and 68° C. for 3minutes; and a final extension at 68° C. for 5 minutes.

The assembled PCR product was gel purified and digested with NdeI andBamHI restriction enzymes. The sites for these restriction enzymes wereintroduced into the assembled PCR products with the OSNBpctF (NdeI) andOSEIBR (BamHI) primers. The digested PCR product was heated at 80° C.for 30 minutes to inactive the restriction enzymes and used directly forligation into a pET11a vector.

The pET-11a vector was digested with NdeI and BamHI restriction enzymes,gel purified using a Qiagen Gel Extraction kit, treated with shrimpalkaline phosphatase (Roche Molecular Biochemicals; Indianapolis, Ind.)and used in a ligation reaction with the assembled PCR product. Theligation was performed at 16° C. overnight using T4 ligase (RocheMolecular Biochemicals; Indianapolis, Ind.). The resulting ligationreaction was transformed into NovaBlue chemically competent cells(Novagen; Madison, Wis.) using a heat-shock method. Once shocked, thecells were plated on LB plates supplemented with 50 μg/mL carbenicillin.The plasmid DNA was purified from individual colonies using a QiaPrepSpin Miniprep Kit (Qiagen Inc., Valencia, Calif.). The resultingplasmids carrying the CoA transferase, 3-hydroxypropionyl-CoAdehydratase, and E1 activator sequences (pTHE1) were digested with XbaIand NdeI, purified using gel electrophoresis and a Qiagen Gel Extractionkit, and used as a vector for cloning of the E2 α subunit/E2 β subunitPCR product.

The E2 α subunit/E2 β subunit PCR product was digested with the sameenzymes and ligated into the pTHE1 vector. The ligation reaction wasperformed at 16° C. overnight using T4 ligase (Roche MolecularBiochemicals, Indianapolis, Ind.). The ligation mixture was transformedinto chemically competent NovaBlue cells (Novagen) that then were platedon LB plates supplemented with 50 μg/mL carbenicillin. The plasmid DNAwas purified from individual colonies using a QiaPrep Spin Miniprep Kit(Qiagen Inc., Valencia, Calif.) and digested with XbaI and NdeIrestriction enzymes for gel electrophoresis analysis. The resultingplasmids carrying the constructed operon #4 (pEIITHEI) were transformedinto BL21(DE3) cells to study the expression of the cloned sequences.Electrospray mass spectrometry assays confirmed that extracts from thesecells have CoA transferase activity and 3-hydroxypropionyl-CoAdehydratase activity. Similar assays are used to confirm that extractsfrom these cells also have lactyl-CoA dehydratase activity.

E. coli plasmid pEIITHrEI carrying a synthetic 3-HP operon was digestedwith NruI, XbaI and BamHI restriction enzymes, XbaI-BamHI DNA fragmentwas gel purified with Quagen Gel Extraction Kit (Qiagen, Inc., ValenciaCalif.) and used for further cloning into Bacillu vector pWH1520(MoBiTec BmBH, Gottingen, Germany). Vector pWH1520 was digested withSpeI and BamHI restriction enzymes and gel purified with Qiagen GelExtraction Kit. The XbaI-BamHI fragment carrying 3-HP operon was ligatedinto WH1520 vector at 16° C. overnight using T4 ligase. The ligationmixture was transformed into chemically competent TOP 10 cells andplated on LB plates supplemented with 50 μg/ml carbenicillin. One clonenamed B. megaterium (pBPO26) was used for assays of CoA-transferase andCoA-hydratase activities. The assays were performed as described abovefor E. Coli. The enzymatic activity was 5 U/mg and 13 U/mg respectively.

Example 7 Construction of a Two Plasmid System

The following constructs were constructed and can be used to produce3-HP in E. coli (FIGS. 38A and B). Nucleic acid molecules encoding a CoAtransferase and a lactyl-CoA dehydratase were amplified from Megasphaeraelsdenii genomic DNA by PCR. Two primers were used to amplify the CoAtransferase-encoding sequence (OSNBpctF and OSHTR), two primers wereused to amplify the E2 α and β subunits of the lactyl-CoAdehydratase-encoding sequence (OSEIIXNF and OSEIIXNR), and two primerswere used to amplify the E1 activator of the lactyl-CoAdehydratase-encoding sequence (E1PROF5′-GTCGCAGAATTCCCATCAATCGCAGCAATCCCAAC-3′, SEQ ID NO:126 and E1PROR5′-TAACATGGTACCGACAGAAGCGGACCAGCAAACGA-3′, SEQ ID NO:127). A nucleicacid molecule encoding a 3-hydroxypropionyl-CoA dehydratase wasamplified from Chloroflexus aurantiacus genomic DNA of by PCR using twoprimers (OSTHF and OSHBR 5′-CGACGGATCCTCAACGACCA-CTGAAGTTGG-3′, SEQ IDNO:128).

PCR was conducted in a Perkin Elmer 2400 Thermocycler using 100 ng ofgenomic DNA and a mix of rTth polymerase (Applied Biosystems; FosterCity, Calif.) and Pfu Turbo polymerase (Stratagene; La Jolla, Calif.) in8:1 ratio. The polymerase mix ensured higher fidelity of the PCRreaction. The following PCR conditions were used: initial denaturationstep of 94° C. for 2 minutes; 20 cycles of 94° C. for 30 seconds, 54° C.for 30 seconds, and 68° C. for 2 minutes; and a final extension at 68°C. for 5 minutes. The obtained PCR products were gel purified using aQiagen Gel Extraction Kit (Qiagen, Inc.; Valencia, Calif.).

The CoA transferase PCR product and the 3-hydroxypropionyl-CoAdehydratase PCR product were assembled using PCR. The OSTHF and OSHTRprimers were complementary to each other. Thus, the complementary DNAends could anneal to each other during the PCR reaction extending theDNA in both direction. To ensure the efficiency of the assembly, two endprimers (OSNBpctF and OSHBR) were added to the assembly PCR mixture,which contained 100 ng of the purified CoA transferase PCR product, 100ng of the purified 3-hydroxypropionyl-CoA dehydratase PCR product, andthe polymerase mix described above. The following PCR conditions wereused to assemble the products: 94° C. for 1 minute; 20 cycles of 94° C.for 30 seconds, 54° C. for 30 seconds, and 68° C. for 2.5 minutes; and afinal extension at 68° C. for 5 minutes.

The assembled PCR product was gel purified and digested with NdeI andBamHI restriction enzymes. The sites for these restriction enzymes wereintroduced into the assembled PCR products with the OSNBpctF (NdeI) andOSHBR (BamHI) primers. The digested PCR product was heated at 80° C. for30 minutes to inactive the restriction enzymes and used directly forligation into a pET11a vector.

The pET-11a vector was digested with NdeI and BamHI restriction enzymes,gel purified using a Qiagen Gel Extraction kit, treated with shrimpalkaline phosphatase (Roche Molecular Biochemicals; Indianapolis, Ind.)and used in a ligation reaction with the assembled PCR product. Theligation was performed at 16° C. overnight using T4 ligase (RocheMolecular Biochemicals; Indianapolis, Ind.). The resulting ligationreaction was transformed into NovaBlue chemically competent cells(Novagen; Madison, Wis.) using a heat-shock method. Once shocked, thecells were plated on LB plates supplemented with 50 μg/mL carbenicillin.The plasmid DNA was purified from individual colonies using a QiaPrepSpin Miniprep Kit (Qiagen Inc.; Valencia, Calif.) and digested with NdeIand BamHI restriction enzymes for gel electrophoresis analysis. Theresulting plasmids carrying the CoA transferase and3-hydroxypropionyl-CoA dehydratase (pTH) were digested with XbaI andNdeI, purified using gel electrophoresis and a Qiagen Gel Extractionkit, and used as a vector for cloning of the E2 α subunit/E2 β subunitPCR product.

The E2 α subunit/E2 β subunit PCR product digested with the same enzymeswas ligated into the pTH vector. The ligation reaction was performed at16° C. overnight using T4 ligase (Roche Molecular Biochemicals;Indianapolis, Ind.). The ligation mixture was transformed intochemically competent NovaBlue cells (Novagen) that then were plated onLB plates supplemented with 50 μg/mL carbenicillin. The plasmid DNA waspurified from individual colonies using a Qiaprep Spin Miniprep Kit(Qiagen Inc.; Valencia, Calif.) and digested with XbaI and NdeIrestriction enzymes for gel electrophoresis analysis. The resultingplasmids carrying the E2 α and β subunits of the lactyl-CoA dehydratase,the CoA transferase, and the 3-hydroxypropionyl-CoA dehydratase (pEIITH)were transformed into BL21(DE3) cells to study the expression of thecloned sequences.

The gel purified E1 activator PCR product was digested with EcoRI andKpnI restriction enzymes, heated at 65° C. for 30 minutes, and ligatedinto a vector (pPROLar.A) that was digested with EcoRI and KpnIrestriction enzymes, gel purified using Qiagen Gel Extraction kit, andtreated with shrimp alkaline phosphatase (Roche Molecular Biochemicals;Indianapolis, Ind.). The ligation was performed at 16° C. overnightusing T4 ligase (Roche Molecular Biochemicals; Indianapolis, Ind.). Theresulting ligation reaction was transformed into DH10B electro-competentcells (Gibco Life Technologies; Gaithersburg, Md.) usingelectroporation. Once electroporated, the cells were plated on LB platessupplemented with 25 μg/mL kanamycin. The plasmid DNA was purified fromindividual colonies using a QiaPrep Spin Miniprep Kit (Qiagen Inc.,Valencia, Calif.) and digested with EcoRI and KpnI restriction enzymesfor gel electrophoresis analysis. The resulting plasmids carrying the E1activator (pPROEI) are transformed into BL21 (DE3) cells to study theexpression of the cloned sequence.

The pPROEI and pEIITH plasmids are compatible plasmids that can be usedin the same bacterial host cell. In addition, the expression from thepPROEI and pEIITH plasmids can be induced at different levels using IPTGand arabinose, allowing for the fine-tuning of the expression of thecloned sequences.

Example 8 Production of 3-HP

3-HP was produced using recombinant E. coli in a small-scale batchfermentation reaction. The construction of strain ALS848 (alsodesignated as TA3476 (J. Bacteriol., 143:1081-1085 (1980))) that carriedinducible T7 RNA polymerase was performed using % DE3 lysogenization kit(Novagen, Madison, Wis.) according to the manufacture's instructions.The constructed strain was designated ALS484(DE3). Strain ALS484(DE3)was transformed with pEIITHrEI plasmid using standard electroporationtechniques. The transformants were selected on LB/carbenicillin (50μg/mL) plates. A single colony was used to inoculate 4 mL culture in a15 mL culture tube. Strain ALS484(DE3) strain carrying vector pET11a wasused as a control. The cells were grown at 37° C. and 250 rpm in anInnova 4230 Incubator Shaker (New Brunswick Scientific, Edison, N.J.)for eight to nine hours. This culture (3 mL) was used to start ananaerobic fermentation. Two 100 mL anaerobic cultures of ALS(DE3)/pET11aand ALS(DE3)pEIITHrEI were grown in serum bottles using LB mediasupplemented with 0.4% glucose, 50 μg/mL carbenicillin, and 100 mM MOPS.The cultures were grown overnight at 37° C. without shaking. Theovernight grown cultures were sub-cultured in serum bottles using freshLB media supplemented with 0.4% glucose, 50 μg/mL carbenicillin, and 100mM MOPS. The starting OD(600) of these cultures was adjusted to 0.3.These serum bottles were incubated at 37° C. without shaking. After onehour of incubation, the cultures were induced with 100 μM IPTG. A 3 mLsample was taken from each of the serum bottles at 30 minutes, 1 hour, 2hours, 4 hours, 6 hours, 8 hours, and 24 hours. The samples weretransferred into two properly labeled 2 mL microcentrifuge tubes, eachcontaining 1.5 mL sample. The samples were spun down in amicrocentrifuge centrifuge at 14000 g for 3 minutes. The supernatant waspassed through a 0.45μ syringe filter, and the resulting filtrate wasstored at −20° C. until further analysis. The formation of fermentationproducts, mainly lactate and 3-hydroxypropionate, was measured bydetecting derivatized CoA esters of lactate and 3-HP using LC/MS.

The following methods were performed to convert lactate and 3-HP intotheir respective CoA esters. Briefly, the filtrates were mixed withCoA-reaction buffer (200 mM potassium phosphate buffer, 2 mM acetyl-CoA,and 0.1 mg/mL purified transferase) in 1:1 ratio. The reaction wasallowed to proceed for 20 minutes at room temperature. The reaction wasstopped by adding 1 volume of 10% TFA. The sample was purified using SepPak Vac columns (Waters). The column was conditioned with methanol andwashed two times with 0.1% TFA. The sample was then applied to thecolumn, and the column was washed two more times with 0.1% TFA. Thesample was eluted with 40% acetonitrile, 0.1% TFA. The acetonitrile wasremoved from the sample by vacuum centrifugation. The samples were thenanalyzed by LC/MS.

Analysis of the standard CoA/CoA thioester mixtures and the CoAthioester mixtures derived from fermentation broths were carried outusing a Waters/Micromass ZQ LC/MS instrument which had a Waters 2690liquid chromatograph with a Waters 996 Photo-Diode Array (PDA)absorbance monitor placed in series between the chromatograph and thesingle quadrupole mass spectrometer. LC separations were made using a4.6×150 mm YMC ODS-AQ (3 μm particles, 120 Å pores) reversed-phasechromatography column at room temperature. Two gradient elution systemswere developed using different mobile phases for the separation of theCoA esters. These two systems are summarized in Table 3. Elution system1 was developed to provide the most rapid and efficient separation ofthe five-component CoA/CoA thioester mixture (CoA, acetyl-CoA,lactyl-CoA, acrylyl-CoA, and propionyl-CoA), while elution system 2 wasdeveloped to provide baseline separation of the structurally isomericesters lactyl-CoA and 3HP-CoA in addition to separation of the remainingesters listed above. In all cases, the flow rate was 0.250 mL/minute,and photodiode array UV absorbance was monitored from 200 nm to 400 nm.All parameters of the electrospray MS system were optimized and selectedbased on generation of protonated molecular ions ([M+H]⁺) of theanalytes of interest and production of characteristic fragment ions. Thefollowing instrumental parameters were used for ESI-MS detection of CoAand organic acid-CoA thioesters in the positive ion mode: Capillary: 4.0V; Cone: 56 V; Extractor: 1 V; RF lens: 0 V; Source temperature: 100°C.; Desolvation temperature: 300° C.; Desolvation gas: 500 L/hour; Conegas: 40 L/hour; Low mass resolution: 13.0; High mass resolution: 14.5;Ion energy: 0.5; Multiplier: 650. Uncertainties for reported mass/chargeratios (m/z) and molecular masses are ±0.01%. Table 3 provides a summaryof gradient elution systems for the separation of organic acid-CoenzymeA thioesters.

TABLE 3 Gradient System Buffer A Buffer B Time Percent B 1 25 mMammonium ACN 0 10 acetate 0.5% acetic acid 0.5% acetic acid 40 40 42 10047 100 50 10 2 25 mM ammonium ACN 0 10 acetate 10 mM TEA 0.5% aceticacid 10 10 0.5% acetic acid 45 60 50 100 53 100 54 10

The following methods were used to separate the derivatized3-hydroxypropionyl-CoA, which was formed from 3-HP, from2-hydroxypropionyl-CoA (i.e., lactyl-CoA), which was formed fromlactate. Because these structural isomers have identical masses and massspectral fragmentation behavior, the separation and identification ofthese analytes in a mixture depends on their chromatographic separation.While elution system 1 provided excellent separation of the CoAthioesters tested (FIG. 46), it was unable to resolve 3-HP-CoA andlactyl-CoA. An alternative LC elution system was developed usingammonium acetate and triethylamine (system 2; Table 3).

The ability of system 2 to separate 3-HP-CoA and lactyl-CoA was testedon a mixture of these two compounds. Comparing the results from amixture of 3-HP-CoA and lactyl-CoA (FIG. 47, Panel A) to the resultsfrom lactyl-CoA only (FIG. 47, Panel B) revealed that system 2 canseparate 3-HP-CoA and lactyl-CoA. The mass spectrum recorded under peak1 (FIG. 47, Panel A insert) was used to identify peak 1 as being ahydroxypropionyl-CoA thioester when compared to FIG. 46, Panel C. Inaddition, comparison of Panels A and B of FIG. 47 as well as the massspectra results corresponding to each peak revealed that peak 1corresponds to 3-HP-CoA and peak 2 corresponds to lactyl-CoA.

System 2 was used to confirm that E. coli transfected with pEIITHrEIproduced 3-HP in that 3-HP-CoA was detected. Specifically, an ionchromatogram for m/z=840 in the analysis of a CoA transferase-treatedfermentation broth aliquot collected from a culture of E. colicontaining pEIITHrEI revealed the presence of 3-HP-CoA (FIG. 48, PanelA). The CoA transferase-treated fermentation broth aliquot collectedfrom a culture of E. coli lacking pEIITHrEI did not exhibit the peakcorresponding to 3-HP-CoA (FIG. 48, Panel B). Thus, these resultsindicate that the pEIITHrEI plasmid directs the expression ofpolypeptides having propionyl-CoA transferase activity, lactyl-CoAdehydratase activity, and acrylyl-CoA hydratase activity. These resultsalso indicate that expression of these polypeptides leads to theformation of 3-HP.

Example 9 Cloning Nucleic Acid Molecules that Encode a PolypeptideHaving Acetyl CoA Carboxylase Activity

Polypeptides having acetyl-CoA carboxylase activity catalyze the firstcommitted step of the fatty acid synthesis by carboxylation ofacetyl-CoA to malonyl-CoA. Polypeptides having acetyl-CoA carboxylaseactivity are also responsible for providing malonyl-CoA for thebiosynthesis of very-long-chain fatty acids required for proper cellfunction. Polypeptides having acetyl-CoA carboxylase activity can bebiotin dependent enzymes in which the cofactor biotin ispost-translationally attached to a specific lysine residue. The reactioncatalyzed by such polypeptides consists of two discrete half reactions.In the first half reaction, biotin is carboxylated by biocarbonate in anATP-dependent reaction to form carboxybiotin. In the second halfreaction, the carboxyl group is transferred to acetyl-CoA to formmalonyl-CoA.

Prokaryotic and eukaryotic polypeptides having acetyl-CoA carboxylaseactivity exist. The prokaryotic polypeptide is a multi-subunit enzyme(four subunits), where each of the subunits is encoded by a differentnucleic acid sequence. For example, in E. coli, the following genesencode for the four subunits of acetyl-CoA carboxylase:

accA: Acetyl-coenzyme a carboxylase carboxyl transferase subunit alpha(GenBank® accession number M96394)

accB: Biotin carboxyl carrier protein (GenBank® accession number U18997)

accC: Biotin carboxylase (GenBank® accession number U18997)

accD: Acetyl-coenzyme a carboxylase carboxyl transferase subunit beta(GenBank® accession number M68934)

The eukaryotic polypeptide is a high molecular weight multi-functionalenzyme encoded by a single gene. For example, in Saccharomycescerevisiae, the acetyl-CoA carboxylase can have the sequence set forthin GenBank® accession number M92156.

The prokaryotic type acetyl-CoA carboxylase from E. coli wasoverexpressed using T7 promoter vector pFN476 as described elsewhere(Davis et al. J. Biol. Chem., 275:28593-28598 (2000)). The eukaryotictype acetyl-CoA carboxylase gene was amplified from Saccharomycescerevisiae genomic DNA. Two primers were designed to amplify the acc1gene from in S. cerevisiae (acc1F5′-atagGCGGCCGCAGGAATGCTGTATGAGCGAAGAAAGCTTATT C-3′, SEQ ID NO: 138where the bold is homologous sequence, the italics is a Not I site, theunderline is a RBS, and the lowercase is extra; and acc1R5′-atgctcgcatCTCGAGTAGCTAAATTAAATTACATCAATAGTA-3′, SEQ ID NO: 139 wherethe bold is homologous sequence, the italics is a Xho I site, and thelowercase is extra). The following PCR mix is used to amplify acc1 gene10×pfu buffer (10 μL), dNTP (10 mM; 2 μL), cDNA (2 μL), acc1F (100 μM; 1μL), acc1R (100 μM; 1 μL), pfu enzyme (2.5 units/μL; 2 μL), and DI water(82 μL). The following protocol was used to amplify the acc1 gene. Afterperforming PCR, the PCR product was separated on a gel, and the bandcorresponding to acc1 nucleic acid (about 6.7 Kb) was gel isolated usingQiagen gel isolation kit. The PCR fragment is digested with Not I andXho I (New England BioLab) restriction enzymes. The digested PCRfragment is then ligated to pET30a which was restricted with Not I andXho I and dephosphorylated with SAP enzyme. The E. coli strain DH10B wastransformed with 1 μL of the ligation mix, and the cells were recoveredin 1 mL of SOC media. The transformed cells were selected onLB/kanamycin (50 μg/μL) plates. Eight single colonies are selected, andPCR was used to screen for the correct insert. The plasmid havingcorrect insert was isolated using Qiagen Spin Mini prep kit.

To obtain a polypeptide having acetyl-CoA carboxylase activity, theplasmid pMSD8 or pET30a/acc1 overexpressing E. coli or S. cerevisiaeacetyl-CoA carboxylase was transformed into Tuner pLacI chemicallycompetent cells (Novagen, Madison, Wis.). The transformed cells wereselected on LB/chloramphenicol (25 μg/mL) plus carbencillin (50 μg/mL)or kanamycin (50 μg/mL).

A crude extract of this strain can be prepared in the following manner.An overnight culture of Tuner pLacI with pMSD8 is subcultured into 200mL (in one liter baffle culture flask) of fresh M9 media supplementedwith 0.4% glucose, 1 μg/mL thiamine, 0.1% casamino acids, and 50 μg/mLcarbencillin or 50 μg/mL kanamycin and 25 μg/mL chloramphenicol. Theculture is grown at 37° C. in a shaker with 250 rpm agitation until itreaches an optical density at 600 nm of about 0.6. IPTG is then added toa final concentration of 100 μM. The culture is then incubated for anadditional 3 hours with shaking speed of 250 rpm at 37° C. Cells areharvested by centrifugation at 8000×g and are washed one time with 0.85%NaCl. The cell pellet was resuspended in a minimal volume of 50 mMTris-HCl (pH 8.0), 5 mM MgCl₂, 100 mM KCl, 2 mM DTT, and 5% glycerol.The cells are lysed by passing them two times through a French Pressurecell at 1000 psig pressure. The cell debris was removed bycentrifugation for 20 minutes at 30,000×g.

The enzyme can be assayed using a method from Davis et al. (J. Biol.Chem., 275:28593-28598 (2000)).

Example 10 Cloning a Nucleic Acid Molecule that Encodes a PolypeptideHaving Malonyl-CoA Reductase Activity from Chloroflexus auarantiacus

A polypeptide having malonyl-CoA reductase activity was partiallypurified from Chloroflexus auarantiacus and used to obtained amino acidmicro-sequencing results. The amino acid sequencing results were used toidentify and clone the nucleic acid that encodes a polypeptide havingmalonyl-CoA reductase activity.

Biomass required for protein purification was grown in B. Braun BIOSTATB fermenters (B. Braun Biotech International GmbH, Melsungen, Germany).A glass vessel fitted with a water jacket for heating was used to growthe required biomass. The glass vessel was connected to its own controlunit. The liquid working volume was 4 L, and the fermenter was operatedat 55° C. with 75 rpm of agitation. Carbon dioxide was occasionallybubbled through the headspace of the fermenter to maintain anaerobicconditions. The pH of the cultures was monitored using a standard pHprobe and was maintained between 8.0 and 8.3. The inoculum for thefermenters was grown in two 250 mL bottles in an Innova 4230 Incubator,Shaker (New Brunswick Scientific, Edison, N.J.) at 55° C. with interiorlights. The fermenters were illuminated by three 65 W plant lightreflector lamps (General Electric, Cleveland, Ohio). Each lamp wasplaced approximately 50 cm away from the glass vessel. The media usedfor the inoculum and the fermenter culture was as follows per liter:0.07 g EDTA, 1 mL micronutrient solution, 1 mL FeCl₃ solution, 0.06 gCaSO₄.2H₂O, 0.1 g MgSO₄.7H₂O, 0.008 g NaCl, 0.075 g KCl, 0.103 g KNO₃,0.68 g NaNO₃, 0.111 g Na₂HPO₄, 0.2 g NH₄Cl, 1 g yeast extract, 2.5 gcasamino acid, 0.5 g Glycyl-Glycine, and 900 mL DI water. Themicronutrient solution contained the following per liter: 0.5 mL H₂SO₄(conc.), 2.28 g MnSO₄.7H₂O, 0.5 g ZnSO₄.7H₂O, 0.5 g H₃BO₃, 0.025 gCuSO₄.2H₂O, 0.025 g Na₂MoO₄.2H₂O, and 0.045 g CoCl₂.6H₂O. The FeCl₃solution contained 0.2905 g FeCl₃ per liter. After adjusting the pH ofthe media to 8.2 to 8.4, 0.75 g/L Na₂S.9H₂O was added, the pH wasreadjusted to 8.2 to 8.4, and the media was filter-sterilized through a0.22μ filter.

The fermenter was inoculated with 500 mL of grown culture. Thefermentation was stopped, and the biomass was harvested after the celldensity was about 0.5 to 0.6 units at 600 nm.

The cells were harvested by centrifugation at 5000×g (Beckman JLA 8.1000rotor) at 4° C., washed with 1 volume of ice cold 0.85% NaCl, andcentrifuged again. The cell pellet was resuspended in 30 mL of ice cold100 mM Tris-HCl (pH 7.8) buffer that was supplemented with 2 mM DTT, 5mM MgCl₂, 0.4 mM PEFABLOC (Roche Molecular Biochemicals, Indianapolis,Ind.), 1% streptomycin sulfate, and 2 tablets of Complete EDTA-freeprotease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis,Ind.). The cell suspension was lysed by passing the suspension, threetimes, through a 50 mL French Pressure Cell operated at 1600 psi (gaugereading). Cell debris was removed by centrifugation at 30,000×g (BeckmanJA 25.50 rotor). The crude extract was filtered prior to chromotographyusing a 0.2 μm HT Tuffryn membrane syringe filter (Pall Corp., AnnArbor, Mich.). The protein concentration of the crude extract was 29mg/mL, which was determined using the BioRad Protein Assay according tothe manufacturer's microassay protocol. Bovine gamma globulin was usedfor the standard curve determination. This assay was based on theBradford dye-binding procedure (Bradford, Anal. Biochem., 72:248(1976)).

Before starting the protein purification, the following assay was usedto determine the activity of malonyl-CoA reductase in the crude extract.A 50 μL aliquot of the cell extract (29 mg/mL) was added to 10 μL 1MTris-HCl (final concentration in assay 100 mM), 10 μL 10 mM malonyl CoA(final concentration in assay 1 mM), 5.5 μL 5.5 mM NADPH (finalconcentration in assay 0.3 mM), and 24.5 μL DI water in a 96 well UVtransparent plate (Corning, N.Y.). The enzyme activity was measured at45° C. using SpectraMAX Plus 96 well plate reader (Molecular devicesSunnyvale, Calif.). The activity of malonyl-CoA reductase was monitoredby measuring the disappearance of NADPH at 340 nm wavelength. The crudeextract exhibited malonyl-CoA reductase activity.

The 5 mL (total 145 mg) protein cell extract was diluted with 20 mLbuffer A (20 mM ethanolamine (pH 9.0), 5 mM MgCl₂, 2 mM DTT). Thechromatographic protein purification was conducted using a BioLogicprotein purification system (BioRad Hercules, Calif.). The 25 mL of cellsuspension was loaded onto a UNO Q-6 ion-exchange column that had beenequilibrated with buffer A at a rate of 1 mL/minute. After sampleloading, the column was washed with 2.5 times column volume of buffer Aat a rate of 2 mL/minute. The proteins were eluted with a lineargradient of NaCl in buffer A from 0-0.33 M in 25 Column volume. Duringthe entire chromatographic separation, three mL fractions werecollected. The collection tubes contained 50 μL of Tris-HCl (pH 6.5) sothat the pH of the eluted sample dropped to about pH 7. Majorchromatographic peaks were detected in the region that corresponded tofractions 18 to 21 and 23 to 30. A 200 μL sample was taken from thesefractions and concentrated in a microcentrifuge at 4° C. using aMicrocon YM-10 columns (Millipore Corp., Bedford, Mass.) as permanufacture's instructions. To each of the concentrated fraction, bufferA-Tris (100 mM Tris-HCl (pH 7.8), 5 mM MgCl₂, 2 mM DTT) was added tobring the total volume to 100 μL. Each of these fractions was tested forthe malonyl-CoA reductase activity using the spectophotometric assaydescribed above. The majority of specific malonyl CoA activity was foundin fractions 18 to 21. These fractions were pooled together, and thepooled sample was desalted using PD-10 column (Amersham PharmaciaPiscataway, N.J.) as per manufacture's instructions.

The 10.5 mL of desalted protein extract was diluted with buffer A-Tristo a volume of 25 mL. This desalted diluted sample was applied to a 1 mLHiTrap Blue column (Amersham Pharmacia Piscataway, N.J.) which wasequilibrated with buffer A-Tris. The sample was loaded at a rate of 0.1mL/minute. Unbound proteins were washed with 2.5 CV buffer A-Tris. Theprotein was eluted with 100 Mm Tris (pH 7.8), 5 mM MgCl₂, 2 mM DTT, 2 mMNADPH, and 1 M NaCl. During this separation process, one mL fractionswere collected. A 200 μL sample was drawn from fractions 49 to 54 andconcentrated. Buffer A-Tris was added to each of the concentratedfractions to bring the total volume to 100 μL. Fractions were assayedfor enzyme activity as described above. The highest specific activitywas observed in fraction 51. The entire fraction 51 was concentrated asdescribed above, and the concentrated sample was separated on anSDS-PAGE gel.

Electrophoresis was carried out using a Bio-Rad Protean II minigelsystem and pre-cast SDS-PAGE gels (4-15%), or a Protean II XI system and16 cm×20 cm×1 mm SDS-PAGE gels (10%) cast as per the manufacturer'sprotocol. The gels were run according to the manufacturer's instructionswith a running buffer of 25 mM Tris-HCl (pH 8.3), 192 mM glycine, and0.1% SDS.

A gel thickness of 1 mm was used to run samples from fraction 51.Protein from fraction 51 was loaded onto 10% SDS-PAGE (3 lanes, eachcontaining 75 μg of total protein). The gels were stained briefly withCoomassie blue (Bio-Rad, Hercules, Calif.) and then destained to a clearbackground with a 10% acetic acid and 20% methanol solution. Thestaining revealed a band of about 130 to 140 KDa.

The protein band of about 130-140 KDa was excised with no excessunstained gel present. An equal area gel without protein was excised asa negative control. The gel slices were placed in uncoloredmicrocentrifuge tubes, prewashed with 50% acetonitrile in HPLC-gradewater, washed twice with 50% acetonitrile, and shipped on dry ice toHarvard Microchemistry Sequencing Facility, Cambridge, Mass.

After in-situ enzymatic digestion of the polypeptide sample withtrypsin, the resulting polypeptides were separated by micro-capillaryreverse-phase HPLC. The HPLC was directly coupled to thenano-electrospray ionization source of a Finnigan LCQ quadrupole iontrap mass spectrometer (μLC/MS/MS). Individual sequence spectra (MS/MS)were acquired on-line at high sensitivity for the multiple polypeptidesseparated during the chromatographic run. The MS/MS spectra of thepolypeptides were correlated with known sequences using the algorithmSequest developed at the University of Washington (Eng et al., J. Am.Soc. Mass Spectrom., 5:976 (1994)) and programs developed at Harvard(Chittum et al., Biochemistry, 37:10866 (1998)). The results werereviewed for consensus with known proteins and for manual confirmationof fidelity.

A similar purification procedure was used to obtain another sample(protein 1 sample) that was subjected to the same analysis that was usedto evaluate the fraction 51 sample.

The polypeptide sequence results indicated that the polypeptidesobtained from both the fraction 51 sample and the protein 1 sample hadsimilarity to the six (764, 799, 859, 923, 1090, 1024) contigs sequencedfrom the C. aurantiacus genome and presented on the Joint GenomeInstitute's web site (http://www.jgi.doe.gov/). The 764 contig was themost prominent of the six with about 40 peptide sequences showingsimilarity. BLASTX analysis of each of these contigs on the GenBank website (http://www.ncbi.nlm.nih.gov/BLAST/) indicated that the DNAsequence of the 764 contig (4201 bases) encoded for polypeptides thathad a dehydrogenase/reductase type activity. Close inspection of the 764contig, however, revealed that this contig did not have an appropriateORF that would encode for a 130-140 KDa polypeptide.

BASLTX analysis also was conducted using the other five contigs. Theresults of this analysis were as follows. The 799 contig (3173 bases)appeared to encode polypeptides having phosphate and dehydrogenase typeactivities. The 859 contig (5865 bases) appeared to encode polypeptideshaving synthetase type activities. The 923 contig (5660 bases) appearedto encode polypeptides having elongation factor and synthetase typeactivities. The 1090 contig (15201 bases) appeared to encodepolypeptides having dehydrogenase/reductase and cytochrome and sigmafactor activities. The 1024 contig (12276 bases) appeared to encodepolypeptides having dehydrogenase and decarboxylase and synthetase typeactivities. Thus, the 859 and 923 contigs were eliminated from anyfurther analysis.

The results from the BLASTX analysis also revealed that thedehydrogenase found in the 1024 contig was most likely an inositolmonophosphate dehydrogenase. Thus, the 1024 contig was eliminated as apossible candidate that might encode for a polypeptide havingmalonyl-CoA reductase activity. The 799 contig also was eliminated sincethis contig is part of the OS17 polypeptide described above.

This narrowed down the search to 2 contigs, the 764 and 1090 contigs.Since the contigs were identified using the same protein sample and thedehydrogenase activities found in these contigs gave very similar BLASTXresults, it was hypothesized that they are part of the same polypeptide.Additional evidence supporting this hypothesis was obtained from thediscovery that the 764 and 1090 contigs are adjacent to each other inthe C. aurantiacus genome as revealed by an analysis of scaffold dataprovided by the Joint Genome Institute. Sequence similarity and assemblyanalysis, however, revealed no overlapping sequence between these twocontigs, possibly due to the presence of gaps in the genome sequencing.

The polypeptide sequences that belonged to the 764 and 1090 contigs weremapped. Based on this analysis, an appropriate coding frame andpotential start and stop codons were identified. The following PCRprimers were designed to PCR amplify a fragment that encoded for apolypeptide having malonyl-CoA reductase activity: PRO140F5′-ATGGCGACGGGCGAGTCCATGAG-3′, SEQ ID NO:153; PRO140R5′-GGACACGAAGAACAGGGCGACAC-3′, SEQ ID NO:154; and PRO140UP5′-GAACTGTCTGGAGTAAGGCTGTC-3′, SEQ ID NO:155. The PRO140F primer wasdesigned based on the sequence of the 1090 contig and corresponds to thestart of the potential start codon. The twelfth base was change from Gto C to avoid primer-dimer formation. This change does not change theamino acid that was encoded by the codon. The PRO140R primer wasdesigned based on sequence of the 764 contig and corresponds to a regionlocated about 1 kB downstream from the potential stop codon. ThePRO140UPF primer was designed based on sequence of the 1090 contig andcorresponds to a region located about 300 bases upstream of potentialstart codon.

Genomic C. aurantiacus DNA was obtained. Briefly, C. aurantiacus wasgrown in 50 mL cultures for 3 to 4 days. Cells were pelleted and washedwith 5 mL of a 10 mM Tris solution. The genomic DNA was then isolatedusing the gram positive bacteria protocol provided with Gentra Genomic“Puregene” DNA isolation kit (Gentra Systems, Minneapolis, Minn.). Thecell pellet was resuspended in 1 mL Gentra Cell Suspension Solution towhich 14.2 mg of lysozyme and 4 μL of 20 mg/mL proteinase K solution wasadded. The cell suspension was incubated at 37° C. for 30 minutes. Theprecipitated genomic DNA was recovered by centrifugation at 3500 g for25 minutes and air-dried for 10 minutes. The genomic DNA was suspendedin an appropriate amount of a 10 mM Tris solution and stored at 4° C.

Two PCR reactions were set-up using C. aurantiacus genomic DNA astemplate as follows:

PCR program PCR Reaction #1 3.3 × rTH polymerase Buffer 30 μL  94° C. 2minutes Mg(OAC) (25 mM) 4 μL 29 cycles of: dNTP Mix (10 mM) 3 μL 94° C.30 seconds PRO140F (100 μM) 2 μL 63° C. 45 seconds PRO140R (100 μM) 2 μL68° C. 4.5 minutes Genomic DNA (100 ng/mL) 1 μL 68° C. 7 minutes rTHpolymerase (2 U/μL) 2 μL  4° C. Until further use pfu polymerase (2.5U/μL) 0.25 μL   DI water 55.75 μL    Total 100 μL  PCR Reaction #2 3.3 ×rTH polymerase Buffer 30 μL  94° C. 2 minutes Mg(OAC) (25 mM) 4 μL 29cycles of: dNTP Mix (10 mM) 3 μL 94° C. 30 seconds PRO140UPF (100 μM) 2μL 60° C. 45 seconds PRO140R (100 μM) 2 μL 68° C. 4.5 minutes GenomicDNA (100 ng/mL) 1 μL 68° C. 7 minutes rTH polymerase (2 U/μL) 2 μL  4°C. Until further use pfu polymerase 2.5 U/μL) 0.25 μL   DI water 55.75μL    Total 100 μL 

The products from both PCR reactions were separated on a 0.8% TAE gel.Both PCR reactions produced a product of 4.7 to 5 Kb in size. Thisapproximately matched the expected size of a nucleic acid molecule thatcould encode a polypeptide having malonyl-CoA reductase activity.

Both PCR products were sequenced using sequencing primers (1090Fseq5′-GATTCCGTATGTCACCCCTA-3′, SEQ ID NO:156; and 764Rseq5′-CAGGCGACTGGCAATCACAA-3′, SEQ ID NO:157). The sequence analysisrevealed a gap between the 764 and 1090 contigs. The nucleic acidsequence between the sequences from the 764 and 1090 contigs was greaterthan 300 base pairs in length (FIG. 51). In addition, the sequenceanalysis revealed an ORF of 3678 bases that showed similarities todehydrogenase/reductase type enzymes (FIG. 52). The amino acid sequenceencoded by this ORF is 1225 amino acids in length (FIG. 50). Also,BLASTP analysis of the amino acid sequence encoded by this ORF revealedtwo short chain dehydrogenase domains (adh type). These results areconsistent with a polypeptide having malonyl-CoA reductase activitysince such an enzyme involves two reduction steps for the conversion ofmalonyl CoA to 3-HP. Further, the computed MW of the polypeptide wasdetermined to be about 134 KDa.

PCR was conducted using the PRO140F/PRO140R primer pair, C. aurantiacusgenomic DNA, and the protocol described above as PCR reaction #1. Afterthe PCR was completed, 0.25 U of Taq polymerase (Roche MolecularBiochemicals, Indianapolis, Ind.) was added to the PCR mix, which wasthen incubated at 72° C. for 10 minutes. The PCR product was columnpurified using Qiagen PCR purification kit (Qiagen Inc., Valencia,Calif.). The purified PCR product was then TOPO cloned into expressionvector pCRT7/CT as per manufacture's instructions (Invitrogen, Carlsbad,Calif.). TOP10 F′ chemical competent cells were transformed with theTOPO ligation mix as per manufacture's instructions (Invitrogen,Carlsbad, Calif.). The cells were recovered for half an hour, and thetransformants were selected on LB/ampicillin (100 μg/mL) plates. Twentysingle colonies were selected, and the plasmid DNA was isolated usingQiagen spin Mini prep kit (Qiagen Inc., Valencia, Calif.).

Each of these twenty clones were tested for correct orientation andright insert size by PCR. Briefly, plasmid DNA was used as a template,and the following two primers were used in the PCR amplification: PCRT75′-GAGACCACAACGGTTTCCCTCTA-3′, SEQ ID NO:158; and PRO140R5′-GGACACGAAGAACAGGGCGACAC-3′, SEQ ID NO:159. The following PCR reactionmix and program was used:

PCR Reaction PCR program 3.3 X rTH polymerase Buffer 7.5 μL 94° C. 2minutes Mg(OAC) (25 mM)   1 μL 25 cycles of: dNTP Mix (10 mM) 0.5 μL 94°C. 30 seconds PCRT7 (100 μM) 0.125 μL  55° C. 45 seconds PRO140R (100μM) 0.125 μL  68° C. 4 minutes Plasmid DNA 0.5 μL 68° C. 7 minutes rTHpolymerase (2 U/μL) 0.5 μL  4° C. Until further use DI water 14.75 μL Total  25 μL

Out of twenty clone tested, only one clone exhibited the correct insert(Clone # P-10). Chemical competent cells of BL21(DE3)pLysS (Invitrogen,Carlsbad, Calif.) were transformed with 2 μL of the P-10 plasmid DNA asper the manufacture's instructions. The cells were recovered at 37° C.for 30 minutes and were plated on LB ampicillin (100 μg/mL) andchloramphenicol (25 μg/mL).

A 20 mL culture of BL21(DE3)pLysS/P-10 and a 20 mL control culture ofBL21(DE3)pLysS was incubated overnight. Using the overnight cultures asan inoculum, two 100 mL BL21(DE3)pLysS/P-10 clone cultures and twocontrol strain cultures (BL21(DE3)pLysS) were started. All the cultureswere induced with IPTG when they reached an OD of about 0.5 at 600 nm.The control strain culture was induced with 10 μM IPTG or 100 μM IPTG,while one of the BL21(DE3)pLysS/P-10 clone cultures was induced with 10μM IPTG and the other with 100 μM IPTG. The cultures were grown for 2.5hours after induction. Aliquots were taken from each of the cultureflasks before and after 2.5 hours of induction and separated using 4-15%SDS-PAGE to analyze polypeptide expression. In the inducedBL21(DE3)pLysS/P-10 samples, a band corresponding to a polypeptidehaving a molecular weight of about 135 KDa was observed. This band wasabsent in the control strain samples and in samples taken before IPTGinduction.

To assess malonyl-CoA reductase activity, BL21(DE3)pLysS/P-10 andBL21(DE3)pLysS cells were cultured and then harvested by centrifugationat 8,000×g (Rotor JA 16.250, Beckman Coulter, Fullerton, Calif.). Onceharvested, the cells were washed once with an equal volume of a 0.85%NaCl solution. The cell pellets were resuspended into 100 mM Tris-HClbuffer that was supplemented with 5 mM Mg₂Cl and 2 mM DTT. The cellswere disrupted by passing twice through a French Press Cell at 1,000 psipressure (Gauge value). The cell debris was removed by centrifugation at30,000×g (Rotor JA 25.50, Beckman Coulter, Fullerton, Calif.). The cellextract was maintained at 4° C. or on ice until further use.

Activity of malonyl-CoA reductase was measured at 37° C. for both thecontrol cells and the IPTG-induced cells. The activity of malonyl-CoAreductase was monitored by observing the disappearance of added NADPH asdescribed above. No activity was found in the cell extract of thecontrol strain, while the cell extract from the IPTG-inducedBL21(DE3)pLysS/P-10 cells displayed malonyl-CoA reductase activity witha specific activity calculated to be about 0.0942 μmole/minute/mg oftotal protein.

Malonyl-CoA reductase activity also was measured by analyzing 3-HPformation from malonyl CoA using the following reaction conducted at 37°C.:

Volume Final conc. Tris HCl (1 M) 10 μL 100 mM  Malonyl CoA (10 mM) 40μL 4 mM NADPH (10 mM) 30 μL 3 mM Cell extract 20 μL Total 100 μL 

The reaction was carried out at 37° C. for 30 minutes. In the controlreaction, a cell extract from BL21(DE3)pLysS was added to a finalconcentration of 322 mg total protein. In the experimental reaction mix,a cell extract from BL21(DE3)pLysS/P-10 was added to a finalconcentration of 226 mg of total protein. The reaction mixtures werefrozen at −20° C. until further analysis.

Chromatographic separation of the components in the reaction mixtureswas performed using a HPX-87H (7.8×300 mm) organic acid HPLC column(BioRad Laboratories, Hercules, Calif.). The column was maintained at60° C. Mobile phase composition was HPLC grade water pH to 2.5 usingtrifluoroacetic acid (TFA) and was delivered at a flow rate of 0.6mL/minute.

Detection of 3-HP in the reaction samples was accomplished using aWaters/Micromass ZQ LC/MS instrument consisting of a Waters 2690 liquidchromatograph (Waters Corp., Milford, Mass.) with a Waters 996Photo-diode Array (PDA) absorbance monitor placed in series between thechromatograph and the single quandrupole mass spectrometer. Theionization source was an Atmospheric Pressure Chemical Ionization (APCI)ionization source. All parameters of the APCI-MS system were optimizedand selected based on the generation of the protonated molecular ion([M+H])⁺ of 3-HP. The following parameters were used to detect 3-HP inthe positive ion mode: Corona: 10 HA; Cone: 20V; Extractor: 2V; RF lens:0.2V; Source temperature: 100° C.; APCI Probe temperature: 300° C.;Desolvation gas: 500 L/hour; Cone gas: 50 L/hour; Low mass resolution:15; High mass resolution: 15; Ion energy: 1.0; Multiplier: 650. Data wascollected in Selected Ion Reporting (SIR) mode set at m/z=90.9.

Both the control reaction sample and the experimental reaction samplewere probed for presence of 3-HP using the HPLC-mass spectroscopytechnique. In the control samples, no 3-HP peak was observed, while theexperimental sample exhibited a peak that matched the retention and themass of 3-HP.

Example 11 Constructing Recombinant Cells that Produce 3-HP

A pathway to make 3-hydroxypropionate directly from glucose via acetylCoA is presented in FIG. 44. Most organisms such as E. coli, Bacillus,and yeast produce acetyl CoA from glucose via glycolysis and the actionof pyruvate dehydrogenase. In order to divert the acetyl CoA generatedfrom glucose, it is desirable to overexpress two genes, one encoding foracetyl CoA carboxylase and the other encoding malonyl-CoA reductase. Asan example, these genes are expressed in E. coli through a T7 promoterusing vectors pET30a and pFN476. The vector pET30a has a pBR ori andkanamycin resistance, while pFN476 has pSC101 ori and uses carbencillinresistance for selection. Because these two vectors have compatible oriand different markers they can be maintained in E. coli at the sametime. Hence, the constructs used to engineer E. coli for directproduction of 3-hydroxypropionate from glucose are pMSD8(pFN476/accABCD) (Davis et al., J. Biol. Chem., 275:28593-28598, 2000)and pET30a/malonyl-CoA reductase or pET30a/acc1 and pFN476/malonyl-CoAreductase. The constructs are depicted in FIG. 45.

To test the production of 3-hydroxypropionate from glucose, E. colistrain Tuner pLacI carrying plasmid pMSD8 (pFN476/accABCD) andpET30a/malonyl-CoA reductase or pET30a/acc1 and pFN476/malonyl-CoAreductase are grown in a B. Braun BIOSTAT B fermenter. A glass vesselfitted with a water jacket for heating is used to conduct thisexperiment. The fermenter working volume is 1.5 L and is operated at 37°C. The fermenter is continuously supplied with oxygen by bubblingsterile air through it at a rate of 1 vvm. The agitation is cascaded tothe dissolve oxygen concentration which is maintained at 40% DO. The pHof the liquid media is maintained at 7 using 2 N NaOH. The E. colistrain is grown in M9 media supplemented with 1% glucose, 1 μg/mLthiamine, 0.1% casamino acids, 10 μg/mL biotin, 50 μg/mL carbencillin,50 μg/mL kanamycin, and 25 μg/mL chloramphenicol. The expression of thegenes is induced when the cell density reached 0.5 OD(600 nm) by adding100 μM IPTG. After induction, samples of 2 mL volume are taken at 1, 2,3, 4, and 8 hours. In addition, at 3 hours after induction, a 200 mLsample is taken to make a cell extract. The 2 mL samples are spun, andthe supernatant is used to analyze products using LC/MS technique. Thesupernatant is stored at −20° C. until further analysis.

The extract is prepared by spinning the 200 mL of cell suspension at8000 g and washing the cell pellet with of 50 mL of 50 mM Tris-HCl (pH8.0), 5 mM MgCl₂, 100 mM KCl, 2 mM DTT, and 5% glycerol. The cellsuspension is spun again at 8000 g, and the pellet is resuspended into 5mL of 50 mM Tris-HCl (pH 8.0), 5 mM MgCl₂, 100 mM KCl, 2 mM DTT, and 5%glycerol. The cells are disrupted by passing twice through a FrenchPress at 1000 pisg. The cell debris is removed by centrifugation for 20minutes at 30,000 g. All the operations are conducted at 4° C. Todemonstrated in vitro formation of 3-hydroxypropionate using thisrecombinant cell extract, the following reaction of 200 μL is conductedat 37° C. The reaction mix is as follows: Tris HCl (pH 8.0; 100 mM), ATP(1 mM), MgCl₂ (5 mM), KCl (100 mM), DTT (5 mM), NaHCO₃ (40 mM), NADPH(0.5 mM), acetyl CoA (0.5 mM), and cell extract (0.2 mg). The reactionis stopped after 15 minutes by adding 1 volume of 10% trifluoroaceticacid (TFA). The products of this reaction are detected using an LC/MStechnique.

The detection and analysis for the presence of 3-hydroxypropionate inthe supernatant and the in vitro reaction mixture is carried out using aWaters/Micromass ZQ LC/MS instrument. This instrument consists of aWaters 2690 liquid chromatograph with a Waters 2410 refractive indexdetector placed in series between the chromatograph and the singlequadropole mass spectrometer. LC separations are made using a Bio-RadAminex 87-H ion-exchange column at 45° C. Sugars, alcohol, and organicacid products are eluted with 0.015% TFA buffer. For elution, the bufferis passed at a flow rate of 0.6 mL/minute. For detection andquantification of 3-hydroxypropionate, a sample obtained from TCI,America (Portland, Oreg.) is used as a standard.

Example 12 Cloning of Propionyl-CoA Transferase, Lactyl-CoA Dehydratase(LDH), and a Hydratase (OS19) for Expression in Saccharomyces cerevisiae

The pESC Yeast Epitope Tagging Vector System (Stratagene, La Jolla,Calif.) was used in cloning the genes involved in 3-hydroxypropionicacid production via lactic acid. The pESC vectors each contain GAL1 andGAL10 promoters in opposing directions, allowing the expression of twogenes from each vector. The GAL1 and GAL10 promoters are repressed byglucose and induced by galactose. Each of the four available pESCvectors has a different yeast-selectable marker (HIS3, TRP1, LEU2, URA3)so multiple plasmids can be maintained in a single strain. Each cloningregion has a polylinker site for gene insertion, a transcriptionterminator, and an epitope coding sequence for C-terminal or N-terminalepitope tagging of expressed proteins. The pESC vectors also have aColE1 origin of replication and an ampicillin resistance gene to allowreplication and selection in E. coli. The followingvector/promoter/nucleic acid combinations were constructed:

Vector Promoter Polypeptide Source of nucleic acid pESC-Trp GAL1 OS19hydratase Chloroflexus aurantiacus GAL10 E1 Megasphaera elsdeniipESC-Leu GAL1 E2α Megasphaera elsdenii GAL10 E2β Megasphaera elsdeniipESC-His GAL1 D-LDH Escherishia coli GAL10 PCT Megasphaera elsdeniiThe primers used were as follows:

OS19APAF: (SEQ ID NO:164) 5′-ATAGGGCCCAGGAGATCAAACCATGGGTGAAGAGTCT-CTGGTT C-3′ OS19SALR: (SEQ IDNO:165) 5′-CCTCTGCTACAGTCGACACAACGACCACTGAAGTTG-GGAG-3′ OS19KPNR: (SEQID NO:166) 5′-AGTCTGCTATCGGTACCTCAACGACCACTGAAGTTG-GGAG-3′ EINOTF: (SEQID NO:167) 5′-ATAGCGGCCGCATAATGGATACTCTCGGAATCGACG-TTGG-3′ EICLAR: (SEQID NO:168) 5′-CCCCATCGATACATATTTCTTGATTTTATCATAAGCA-ATC-3′ EIIαAPAF:(SEQ ID NO:169) 5′-CCAGGGCCCATAATGGGTGAAGAAAAAACAGTAGA-TATTG-3′EIIαSALR: (SEQ ID NO:170) 5′-GGTAGACTTGTCGACGTAGTGGTTTCCTCCTTCATT-GG-3′EIIβNOTF: (SEQ ID NO:171)5′-ATAGCGGCCGCATAATGGGTCAGATCGACGAACTTA-TCAG-3′ EIIβSPER: (SEQ IDNO:172) 5′-AGGTTCAACTAGTTCGTAGAGGATTTCCGAGAAAGC-CTG-3′ LDHAPAF: (SEQ IDNO:173) 5′-CTAGGGCCCATAATGGAACTCGCCGTTTATAG-CAC-3′ LDHXHOR: (SEQ IDNO:174) 5′-ACTTCTCGAGTTAAACCAGTTCGTTCGGGCA-GGT-3′ PCTSPEF: (SEQ IDNO:175) 5′-GGGACTAGTATAATGGGAAAAGTAGAAATCAT-TACAG-3′ PCTPACR: (SEQ IDNO:176) 5′-CGGCTTAATTAACAGCAGAGATTTATTTTTTCA-GTCC-3′

All restriction enzymes were obtained from New England Biolabs, Beverly,Mass. All plasmid DNA preparations were done using QIAprep Spin MiniprepKits, and all gel purifications were done using QIAquick Gel ExtractionKits (Qiagen, Valencia, Calif.).

A. Construction of the pESC-Trp/OS19 Hydratase Vector

Two constructs in pESC-Trp were made for the OS19 nucleic acid from C.aurantiacus. One of these constructs utilized the Apa I and Sal Irestriction sites of the GAL1 multiple cloning site and was designed toinclude the c-myc epitope. The second construct utilized the Apa I andKpn I sites and thus did not include the c-myc epitope sequence.

Six μg of pESC-Trp vector DNA was digested with the restriction enzymeApa I and the digest was purified using a QIAquick PCR PurificationColumn. Three μg of the Apa I-digested vector DNA was then digested withthe restriction enzyme Kpn I, and 3 μg was digested with Sal I. Thedouble-digested vector DNAs were separated on a 1% TAE-agarose gel,purified, dephosphorylated with shrimp alkaline phosphatase (RocheBiochemical Products, Indianapolis, Ind.), and purified with a QIAquickPCR Purification Column.

The nucleic acid encoding the Chloroflexus aurantiacus polypeptidehaving hydratase activity (OS19) was amplified from genomic DNA usingthe PCR primer pair OS19APAF and OS19SALR and the primer pair OS19APAFand OS19 KPNR. OS19APAF was designed to introduce an Apa I restrictionsite and a translation initiation site (ACCATGG) at the beginning of theamplified fragment. The OS19 KPNR primer was designed to introduce a KpnI restriction site at the end of the amplified fragment and to containthe translational stop codon for the hydratase gene. OS19SALR introducesa Sal I site at the end of the amplified fragment and has an alteredstop codon so that translation continues in-frame through the vectorc-myc epitope. The PCR mix contained the following: 1× Expand PCRbuffer, 100 ng C. aurantiacus genomic DNA, 0.2 μM of each primer, 0.2 mMeach dNTP, and 5.25 units of Expand DNA Polymerase (Roche) in a finalvolume of 100 μL. The PCR reaction was performed in an MJ ResearchPTC100 under the following conditions: an initial denaturation at 94° C.for 1 minute; 8 cycles of 94° C. for 30 seconds, 57° C. for 1 minute,and 72° C. for 2.25 minutes; 24 cycles of 94° C. for 30 seconds, 62° C.for 1 minute, and 72° C. for 2.25 minutes; and a final extension for 7minutes at 72° C. The amplification product was then separated by gelelectrophoresis using a 1% TAE-agarose gel. A 0.8 Kb fragment wasexcised from the gel and purified for each primer pair. The purifiedfragments were digested with Kpn I or Sal I restriction enzyme, purifiedwith a QIAquick PCR Purification Column, digested with Apa I restrictionenzyme, purified again with a QIAquick PCR Purification Column, andquantified on a minigel.

50-60 ng of the digested PCR product containing the nucleic acidencoding the C. aurantiacus polypeptide having hydratase activity (OS19)and 50 ng of the prepared pESC-Trp vector were ligated using T4 DNAligase at 16° C. for 16 hours. One μL of the ligation reaction was usedto electroporate 40 μL of E. coli Electromax™ DH10B™ cells. Theelectroporated cells were plated onto LB plates containing 100 μg/mL ofcarbenicillin (LBC). Individual colonies were screened using colony PCRwith the appropriate PCR primers. Individual colonies were suspended inabout 25 μL of 10 mM Tris, and 2 μL of the suspension was plated on LBCmedia. The remnant suspension was heated for 10 minutes at 95° C. tobreak open the bacterial cells, and 2 μL of the heated cells was used ina 25 μL PCR reaction. The PCR mix contained the following: 1×Taq buffer,0.2 μM each primer, 0.2 mM each dNTP, and 1 unit of Taq DNA polymeraseper reaction. The PCR program used was the same as described above foramplification of the nucleic acid from genomic DNA.

Plasmid DNA was isolated from cultures of colonies having the desiredinsert and was sequenced to confirm the lack of nucleotide errors fromPCR. A construct with a confirmed sequence was transformed into S.cerevisiae strain YPH500 using a Frozen-EZ Yeast Transformation II™ Kit(Zymo Research, Orange, Calif.). Transformation reactions were plated onSC-Trp media (see Stratagene pESC Vector Instruction Manual for mediarecipes). Individual yeast colonies were screened for the presence ofthe OS19 nucleic acid by colony PCR. Colonies were suspended in 20 μL ofY-Lysis Buffer (Zymo Research) containing 5 units of zymolase and heatedat 37° C. for 10 minutes. Three μL of this suspension was then used in a25 μL PCR reaction using the PCR reaction mixture and program describedfor the colony screen of the E. coli transformants. The pESC-Trp vectorwas also transformed into YPH500 for use as a hydratase assay controland transformants were screened by PCR using GAL1 and GAL10 primers.

B. Construction of the pESC-Trp/OS19/EI Hydratase Vector

Plasmid DNA of a pESC-Trp/OS19 construct (Apa I-Sal I sites) withconfirmed sequence and positive assay results was used for insertion ofthe nucleic acid for the M. elsdenii E1 activator polypeptide downstreamof the GAL10 promoter. Three μg of plasmid DNA was digested with therestriction enzyme Cla I, and the digest was purified using a QIAquickPCR Purification Column. The vector DNA was then digested with therestriction enzyme Not I, and the digest was inactivated by heating to65° C. for 20 minutes. The double-digested vector DNA wasdephosphorylated with shrimp alkaline phosphatase (Roche), separated ona 1% TAE-agarose gel, and gel purified.

The nucleic acid encoding the M. elsdenii E1 activator polypeptide wasamplified from genomic DNA using the PCR primer pair EINOTF and EICLAR.EINOTF was designed to introduce a Not I restriction site and atranslation initiation site at the beginning of the amplified fragment.The EICLAR primer was designed to introduce a Cla I restriction site atthe end of the amplified fragment and to contain an alteredtranslational stop codon to allow in-frame translation of the FLAGepitope. The PCR mix contained the following: 1× Expand PCR buffer, 100ng M. elsdenii genomic DNA, 0.2 μM of each primer, 0.2 mM each dNTP, and5.25 units of Expand DNA Polymerase in a final volume of 100 μL. The PCRreaction was performed in an MJ Research PTC100 under the followingconditions: an initial denaturation at 94° C. for 1 minute; 8 cycles of94° C. for 30 seconds, 55° C. for 45 seconds, and 72° C. for 3 minutes;24 cycles of 94° C. for 30 seconds, 62° C. for 45 seconds, and 72° C.for 3 minutes; and a final extension for 7 minutes at 72° C. Theamplification product was then separated by gel electrophoresis using a1% TAE-agarose gel, and a 0.8 Kb fragment was excised and purified. Thepurified fragment was digested with Cla I restriction enzyme, purifiedwith a QIAquick PCR Purification Column, digested with Not I restrictionenzyme, purified again with a QIAquick PCR Purification Column, andquantified on a minigel.

60 ng of the digested PCR product containing the nucleic acid for the M.elsdenii E1 activator polypeptide and 70 ng of the preparedpESC-Trp/OS19 hydratase vector were ligated using T4 DNA ligase at 16°C. for 16 hours. One μL of the ligation reaction was used toelectroporate 40 μL of E. coli Electromax™ DH10B™ cells. Theelectroporated cells were plated onto LBC media. Individual colonieswere screened using colony PCR with the EINOTF and EICLAR primers.Individual colonies were suspended in about 25 μL of 10 mM Tris, and 2μL of the suspension was plated on LBC media. The remnant suspension washeated for 10 minutes at 95° C. to break open the bacterial cells, and 2μL of the heated cells used in a 25 μL PCR reaction. The PCR mixcontained the following: 1×Taq buffer, 0.2 μM each primer, 0.2 mM eachdNTP, and 1 unit of Taq DNA polymerase per reaction. The PCR programused was the same as described above for amplification of the gene fromgenomic DNA. Plasmid DNA was isolated from cultures of colonies havingthe desired insert and was sequenced to confirm the lack of nucleotideerrors from PCR.

C. Construction of the pESC-Leu/EIIα/EIIβ Vector

Three μg of DNA of the vector pESC-Leu was digested with the restrictionenzyme Apa I, and the digest was purified using a QIAquick PCRPurification Column. The vector DNA was then digested with therestriction enzyme Sal I, and the digest was inactivated by heating to65° C. for 20 minutes. The double-digested vector DNA wasdephosphorylated with shrimp alkaline phosphatase (Roche), separated ona 1% TAE-agarose gel, and gel purified.

The nucleic acid encoding the M. elsdenii E2α polypeptide was amplifiedfrom genomic DNA using the PCR primer pair EIIαAPAF and EIIaαSALR.EIIαAPAF was designed to introduce an Apa I restriction site and atranslation initiation site at the beginning of the amplified fragment.The EIIαSALR primer was designed to introduce a Sal I restriction siteat the end of the amplified fragment and to contain an alteredtranslational stop codon to allow in-frame translation of the c-mycepitope. The PCR mix contained the following: 1× Expand PCR buffer, 100ng M. elsdenii genomic DNA, 0.2 μM of each primer, 0.2 mM each dNTP, and5.25 units of Expand DNA Polymerase in a final volume of 100 μL. The PCRreaction was performed in an MJ Research PTC100 under the followingconditions: an initial denaturation at 94° C. for 1 minute; 8 cycles of94° C. for 30 seconds, 55° C. for 1 minute, and 72° C. for 3 minutes; 24cycles of 94° C. for 30 seconds, 62° C. for 1 minute, and 72° C. for 3minutes; and a final extension for 7 minutes at 72° C. The amplificationproduct was then separated by gel electrophoresis using a 1% TAE-agarosegel, and a 1.3 Kb fragment was excised and purified. The purifiedfragment was digested with Apa I restriction enzyme, purified with aQIAquick PCR Purification Column, digested with Sal I restrictionenzyme, purified again with a QIAquick PCR Purification Column, andquantified on a minigel.

80 ng of the digested PCR product containing the nucleic acid encodingthe M. elsdenii E2α polypeptide and 80 ng of the prepared pESC-Leuvector were ligated using T4 DNA ligase at 16° C. for 16 hours. One μLof the ligation reaction was used to electroporate 40 μL of E. coliElectromax™ DH10B™ cells. The electroporated cells were plated onto LBCmedia. Individual colonies were screened using colony PCR with theEIIαAPAF and EIIαSALR primers. Individual colonies were suspended inabout 25 μl of 10 mM Tris, and 2 μL of the suspension was plated on LBCmedia. The remnant suspension was heated for 10 minutes at 95° C. tobreak open the bacterial cells, and 2 μL of the heated cells used in a25 μL PCR reaction. The PCR mix contained the following: 1×Taq buffer,0.2 μM each primer, 0.2 mM each dNTP, and 1 unit of Taq DNA polymeraseper reaction. The PCR program used was the same as described above foramplification of the gene from genomic DNA. Plasmid DNA was isolatedfrom cultures of colonies having the desired insert and was sequenced toconfirm the lack of nucleotide errors from PCR.

Plasmid DNA of a pESC-Leu/EIIα vector with confirmed sequence was usedfor insertion of the nucleic acid encoding the M. elsdenii E2βpolypeptide. Three μg of plasmid DNA was digested with the restrictionenzyme Spe I, and the digest was purified using a QIAquick PCRPurification Column. The vector DNA was then digested with therestriction enzyme Not I and gel purified from a 1% TAE-agarose gel. Thedouble-digested vector DNA was then dephosphorylated with shrimpalkaline phosphatase (Roche) and purified with a QIAquick PCRPurification Column.

The nucleic acid encoding the M. elsdenii E2β polypeptide was amplifiedfrom genomic DNA using the PCR primer pair EIIβNOTF and EIIβSPER. TheEIIβNOTF primer was designed to introduce a Not I restriction site and atranslation initiation site at the beginning of the amplified fragment.The EIIβSPER primer was designed to introduce an Spe I restriction siteat the end of the amplified fragment and to contain an alteredtranslational stop codon to allow for in-frame translation of the FLAGepitope. The PCR mix contained the following: 1× Expand PCR buffer, 100ng M. elsdenii genomic DNA, 0.2 μM of each primer, 0.2 mM each dNTP, and5.25 units of Expand DNA Polymerase in a final volume of 100 μL. The PCRreaction was performed in an MJ Research PTC100 under the followingconditions: an initial denaturation at 94° C. for 1 minute; 8 cycles of94° C. for 30 seconds, 55° C. for 45 seconds, and 72° C. for 3 minutes;24 cycles of 94° C. for 30 seconds, 62° C. for 45 seconds, and 72° C.for 3 minutes; and a final extension for 7 minutes at 72° C. Theamplification product was separated by gel electrophoresis using a 1%TAE-agarose gel, and a 1.1 Kb fragment was excised and purified. Thepurified fragment was digested with Spe I restriction enzyme, purifiedwith a QIAquick PCR Purification Column, digested with Not I restrictionenzyme, purified again with a QIAquick PCR Purification Column, andquantified on a minigel.

38 ng of the digested PCR product containing the nucleic acid encodingthe M. elsdenii E2β polypeptide and 50 ng of the prepared pESC-Leu/EIIαvector were ligated using T4 DNA ligase at 16° C. for 16 hours. One μLof the ligation reaction was used to electroporate 40 μL of E. coliElectromax™ DH10B™ cells. The electroporated cells were plated onto LBCplates. Individual colonies were screened using colony PCR with theEIIβNOTF and EIIβSPER primers. Individual colonies were suspended inabout 25 μL of 10 mM Tris, and 2 μL of the suspension was plated on LBCmedia. The remnant suspension was heated for 10 minutes at 95° C. tobreak open the bacterial cells, and 2 μL of the heated cells was used ina 25 μL PCR reaction. The PCR mix contained the following: 1×Taq buffer,0.2 μM each primer, 0.2 mM each dNTP, and 1 unit of Taq DNA polymeraseper reaction. The PCR program used was the same as described above foramplification of the gene from genomic DNA.

Plasmid DNA was isolated from cultures of colonies having the desiredinsert and was sequenced to confirm the lack of nucleotide errors fromPCR. A pESC-Leu/EIIα/EIIβ construct with a confirmed sequence wasco-transformed along with the pESC-Trp/OS19/EI vector into S. cerevisiaestrain YPH500 using a Frozen-EZ Yeast Transformation II™ Kit (ZymoResearch, Orange, Calif.). Transformation reactions were plated onSC-Trp-Leu media. Individual yeast colonies were screened for thepresence of the OS19, E1, E2α, and E2β nucleic acid by colony PCR.Colonies were suspended in 20 μL of Y-Lysis Buffer (Zymo Research)containing 5 units of zymolase and heated at 37° C. for 10 minutes.Three μL of this suspension was then used in a 25 μL PCR reaction usingthe PCR reaction mixtures and programs described for the colony screensof the E. coli transformants. The pESC-Trp/OS19 and pESC-Leu vectorswere also co-transformed into YPH500 for use as a lactyl-CoA dehydrataseassay control. These transformants were colony screened using the GAL1and GAL10 primers (Instruction manual, pESC Yeast Epitope TaggingVectors, Stratagene).

D. Construction of the pESC-His/D-LDH/PCT Vector

Three μg of DNA of the vector pESC-His was digested with the restrictionenzyme Xho I, and the digest was purified using a QIAquick PCRPurification Column. The vector DNA was then digested with therestriction enzyme Apa I and gel purified from a 1% TAE-agarose gel. Thedouble-digested vector DNA was dephosphorylated with shrimp alkalinephosphatase (Roche) and purified using a QIAquick PCR PurificationColumn.

The E. coli D-LDH gene was amplified from genomic DNA of strain DH10Busing the PCR primer pair LDHAPAF and LDHXHOR. LDHAPAF was designed tointroduce an Apa I restriction site and a translation initiation site atthe beginning of the amplified fragment. The LDHXHOR primer was designedto introduce an Xho I restriction site at the end of the amplifiedfragment and to contain the translational stop codon for the D-LDH gene.The PCR mix contained the following: 1× Expand PCR buffer, 100 ng E.coli genomic DNA, 0.2 μM of each primer, 0.2 mM each dNTP, and 5.25units of Expand DNA Polymerase in a final volume of 100 μL. The PCRreaction was performed in an MJ Research PTC100 under the followingconditions: an initial denaturation at 94° C. for 1 minute; 8 cycles of94° C. for 30 seconds, 59° C. for 45 seconds, and 72° C. for 2 minutes;24 cycles of 94° C. for 30 seconds, 64° C. for 45 seconds, and 72° C.for 2 minutes; and a final extension for 7 minutes at 72° C. Theamplification product was separated by gel electrophoresis using a 1%TAE-agarose gel, and a 1.0 Kb fragment was excised and purified. Thepurified fragment was digested with Apa I restriction enzyme, purifiedwith a QIAquick PCR Purification Column, digested with Xho I restrictionenzyme, purified again with a QIAquick PCR Purification Column, andquantified on a minigel.

80 ng of the digested PCR product containing the E. coli D-LDH gene and80 ng of the prepared pESC-His vector were ligated using T4 DNA ligaseat 16° C. for 16 hours. One μL of the ligation reaction was used toelectroporate 40 μL of E. coli Electromax™ DH10B™ cells. Theelectroporated cells were plated onto LBC media. Individual colonieswere screened using colony PCR with the LDHAPAF and LDHXHOR primers.Individual colonies were suspended in about 25 μL of 10 mM Tris, and 2μL of the suspension was plated on LBC media. The remnant suspension washeated for 10 minutes at 95° C. to break open the bacterial cells, and 2μL of the heated cells used in a 25 μL PCR reaction. The PCR mixcontained the following: 1×Taq buffer, 0.2 μM each primer, 0.2 mM eachdNTP, and 1 unit of Taq DNA polymerase per reaction. The PCR programused was the same as described above for amplification of the gene fromgenomic DNA. Plasmid DNA was isolated from cultures of colonies havingthe desired insert and was sequenced to confirm the lack of nucleotideerrors from PCR.

Plasmid DNA of a pESC-His/D-LDH construct with a confirmed sequence wasused for insertion of the nucleic acid encoding the M. elsdenii PCTpolypeptide. Three μg of plasmid DNA was digested with the restrictionenzyme Pac I, and the digest was purified using a QIAquick PCRPurification Column. The vector DNA was then digested with therestriction enzyme Spe I and gel purified from a 1% TAE-agarose gel. Thedouble-digested vector DNA was dephosphorylated with shrimp alkalinephosphatase (Roche) and purified with a QIAquick PCR PurificationColumn.

The nucleic acid encoding the M. elsdenii PCT polypeptide was amplifiedfrom genomic DNA using the PCR primer pair PCTSPEF and PCTPACR. PCTSPEFwas designed to introduce an Spe I restriction site and a translationinitiation site at the beginning of the amplified fragment. The PCTPACRprimer was designed to introduce a Pac I restriction site at the end ofthe amplified fragment and to contain the translational stop codon forthe PCT gene. The PCR mix contained the following: 1×Expand PCR buffer,100 ng M. elsdenii genomic DNA, 0.2 μM of each primer, 0.2 mM each dNTP,and 5.25 units of Expand DNA Polymerase in a final volume of 100 μL. ThePCR reaction was performed in an MJ Research PTC100 under the followingconditions: an initial denaturation at 94° C. for 1 minute; 8 cycles of94° C. for 30 seconds, 56° C. for 45 seconds, and 72° C. for 2.5minutes; 24 cycles of 94° C. for 30 seconds, 64° C. for 45 seconds, and72° C. for 2.5 minutes; and a final extension for 7 minutes at 72° C.The amplification product was separated by gel electrophoresis using a1% TAE-agarose gel, and a 1.55 Kb fragment was excised and purified. Thepurified fragment was digested with Pac I restriction enzyme, purifiedwith a QIAquick PCR Purification Column, digested with Spe I restrictionenzyme, purified again with a QIAquick PCR Purification Column, andquantified on a minigel.

95 ng of the digested PCR product containing the nucleic acid encodingthe M. elsdenii PCT polypeptide and 75 ng of the prepared pESC-His/D-LDHvector were ligated using T4 DNA ligase at 16° C. for 16 hours. One μLof the ligation reaction was used to electroporate 40 μL of E. coliElectromax™ DH10B™ cells. The electroporated cells were plated onto LBCplates. Individual colonies were screened using colony PCR with thePCTSPEF and PCTPACR primers. Individual colonies were suspended in about25 μL of 10 mM Tris, and 2 μL of the suspension was plated on LBC media.The remnant suspension was heated for 10 minutes at 95° C. to break openthe bacterial cells, and 2 μL of the heated cells used in a 25 μL PCRreaction. The PCR mix contained the following: 1×Taq buffer, 0.2 μM eachprimer, 0.2 mM each dNTP, and 1 unit of Taq DNA polymerase per reaction.The PCR program used was the same as described above for amplificationof the gene from genomic DNA.

Plasmid DNA was isolated from cultures of colonies having the desiredinsert and was sequenced to confirm the lack of nucleotide errors fromPCR. A construct with a confirmed sequence was transformed into S.cerevisiae strain YPH500 using a Frozen-EZ Yeast Transformation II™ Kit(Zymo Research, Orange, Calif.). Transformation reactions were plated onSC-His media. Individual yeast colonies were screened for the presenceof the D-LDH and PCT genes by colony PCR. Colonies were suspended in 20μL of Y-Lysis Buffer (Zymo Research) containing 5 units of zymolase andheated at 37° C. for 10 minutes. Three μL of this suspension was thenused in a 25 μL PCR reaction using the PCR reaction mixture and programdescribed for the colony screen of the E. coli transformants. ThepESC-His vector was also transformed into YPH500 for use as an assaycontrol, and transformants were screened by PCR using GAL1 and GAL10primers.

Example 13 Expression of Enzymes in S. cerevisiae A. Hydratase Activityin Transformed Yeast

Individual colonies carrying the pESC-Trp/OS19 construct or the pESC-Trpvector (negative control) were used to inoculate 5 mL cultures of SC-Trpmedia containing 2% glucose. These cultures were grown for 16 hours at30° C. and used to inoculate 35 mL of the same media. The subcultureswere grown for 7 hours at 30° C., and their OD₆₀₀s were determined. Avolume of cells giving an OD×volume equal to 40 was pelleted, washedwith SC-Trp media with no carbon source, and repelleted. The cells weresuspended in 5 mL of SC-Trp media containing 2% galactose and used toinoculate a total volume of 100 mL of this media. Cultures were grownfor 17.5 hours at 30° C. and 250 rpm. Cells were then pelleted, rinsedin 0.85% NaCl, and repelleted. Cell pellets (70 mg) were suspended in140 μL of 50 mM Tris HCl, pH 7.5, and an equal volume (pellet plusbuffer) of pre-rinsed glass beads (Sigma, 150-212 microns) was added.This mixture was vortexed for 30 seconds and placed on ice for 1 minute,and the vortexing/cooling cycle was repeated 8 additional times. Thecells were then centrifuged for 6 minutes at 5,000 g, and thesupernatant was removed to a fresh tube. The beads/pellet were washedtwice with 250 μL of buffer, centrifuged, and the supernatants joinedwith the first supernatant.

An E. coli strain carrying the pETBlue-1/OS19 construct, describedpreviously, was used as a positive control for hydratase assays. Aculture of this strain was grown to saturation overnight and diluted1:20 the following morning in fresh LBC media. The culture was grown at37° C. and 250 rpm to an OD₆₀₀ of 0.6, at which point it was inducedwith IPTG at a final concentration of 1 mM. The culture was incubatedfor an additional two hours at 37° C. and 250 rpm. Cells were pelleted,washed with 0.85% NaCl, and repelleted. Cells were disrupted usingBugBuster™ Protein Extraction Reagent and Benzonase® (Novagen) as permanufacturer's instructions with a 20 minute incubation at roomtemperature. After centrifugation at 16,000 g and 4° C., the supernatantwas transferred to a new tube and used in the activity assay.

Total protein content of cell extracts from S. cerevisiae describedabove were quantified using a microplate Bio-Rad Protein Assay (Bio-Rad,Hercules, Calif.). The OS19 constructs (both Apa I-Sal I and Apa I-Kpn Isites) in YPH500, the pESC-Trp negative control in YPH500, and thepETBlue-1/OS19 construct in E. coli were tested for their ability toconvert acrylyl-CoA to 3-hydroxypropionyl-CoA. The assay was conductedas previously described for the pETBlue-1/OS19 constructs in the E. coliTuner strain. When cell extract of the negative control strain was addedto the reaction mixture containing acrylyl-CoA, one dominant peak of MW823 was exhibited. This peak corresponds to acrylyl-CoA and indicatesthat acrylyl-CoA was not converted to any other product. When cellextract of the strain carrying a pESC-Trp/OS19 construct (either ApaI-Sal I or Apa I-Kpn I sites) was added to the reaction mix, thedominant peak shifted to MW 841, which corresponds to3-hydroxypropionyl-CoA. The reaction mix from the E. coli control alsoshowed the MW 841 peak. A time course study was conducted for thepESC-Trp/OS19(Apa I-Sal I) construct, which measured the appearance ofthe MW 841 and MW 823 peaks after 0, 1, 3, 7, 15, 30, and 60 minutes ofreaction time. An increase in the 3-hydroxypropionyl-CoA peak was seenover time with the cell extracts from both this construct and the E.coli control, whereas cell extract from the YPH500 strain with vectoronly showed a dominant acrylyl-CoA peak.

B. Propionyl CoA-Transferase Activity in Transformed Yeast

Individual colonies of S. cerevisiae strain YPH500 carrying thepESC-His/D-LDH or pESC-His/D-LDH/PCT construct or the pESC-His vectorwith no insert (negative control) were used to inoculate 5 mL culturesof SC-His media containing 2% glucose. These cultures were grown for 16hours at 30° C. and 250 rpm and were then used to inoculate 35 mL of thesame media. The subcultures were grown for 7 hours at 30° C., and theirOD₆₀₀s were determined. For each strain, a volume of cells giving anOD×volume equal to 40 was pelleted, washed with SC-His media with nocarbon source, and repelleted. The cells were suspended in 5 mL ofSC-His media containing 2% galactose and used to inoculate a totalvolume of 100 mL of this media. Cultures were grown for 16.75 hours at30° C. and 250 rpm. Cells were then pelleted, rinsed in 0.85% NaCl, andrepelleted. Cell pellets (70 mg) were suspended in 140 μL of 100 mMpotassium phosphate buffer, pH 7.5, and an equal volume (pellet plusbuffer) of pre-rinsed glass beads (Sigma, 150-212 microns) was added.This mixture was vortexed for 30 seconds and placed on ice for 1 minute,and the vortexing/cooling cycle was repeated 8 additional times. Thecells were then centrifuged for 6 minutes at 5,000 g, and thesupernatant was removed to a fresh tube. The beads/pellet were washedtwice with 250 μL of buffer and centrifuged, and the supernatants joinedwith the first supernatant.

An E. coli strain carrying the pETBlue-1/PCT construct, describedpreviously, was used as a positive control for propionyl CoA transferaseassays. A culture of this strain was grown to saturation overnight anddiluted 1:20 the following morning in fresh LB media containing 100μg/mL of carbenicillin. The culture was grown at 37° C. and 250 rpm toan OD₆₀₀ of 0.6, at which point it was induced with IPTG at a finalconcentration of 1 mM. The culture was incubated for an additional twohours at 37° C. and 250 rpm. Cells were pelleted, washed with 0.85%NaCl, and repelleted. Cells were disrupted using BugBuster™ ProteinExtraction Reagent and Benzonase® (Novagen) as per manufacturer'sinstructions with a 20 minute incubation at room temperature. Aftercentrifugation at 16,000 g and 4° C., the supernatant was transferred toa new tube and used in the activity assay.

Total protein content of cell extracts was quantified using a microplateBio-Rad Protein Assay (Bio-Rad, Hercules, Calif.). The D-LDH andD-LDH/PCT constructs in S. cerevisiae strain YPH500, the pESC-Hisnegative control in YPH500, and the pETBlue-1/PCT construct in E. coliwere tested for their ability to catalyze the conversion ofpropionyl-CoA and acetate to acetyl-CoA and propionate. The assaymixture used was that previously described for the pETBlue-1/PCTconstructs in the E. coli Tuner strain.

When 1 μg of total cell extract protein of the negative control strainor the YPH500/pESC-His/D-LDH strain was added to the reaction mixture,no increase in absorbance (0.00 to 0.00) was seen over 11 minutes.Increases in absorbance from 0.00 to 0.04 and from 0.00 to 0.06 wereseen, respectively, with 1 μg of cell extract protein from theYPH500/pESC-His/D-LDH/PCT strain and the E. coli/PCT strain. With 2 mgof total cell extract protein, the negative control strain and theYPH500/pESC-His/D-LDH strain showed an increase in absorbance from 0.00to 0.01 over 11 minutes, whereas increases from 0.00 to 0.10 and 0.00 to0.08 were seen, respectively, with the YPH500/pESC-His/D-LDH/PCT strainand the E. coli/PCT strain.

C. Lactyl-CoA Dehvdratase Activity in Transformed Yeast

Individual colonies of S. cerevisiae strain YPH500 carrying thepESC-His/D-LDH or pESC-His/D-LDH/PCT construct or the pESC-His vectorwith no insert (negative control) were used to inoculate 5 mL culturesof SC-His media containing 4% glucose. These cultures were grown for 23hours at 30° C. and used to inoculate 35 mL of SC-His media containing2% raffinose. The subcultures were grown for 8 hours at 30° C., andtheir OD₆₀₀s were determined. For each strain, a volume of cells givingan OD×volume equal to 40 was pelleted, resuspended in 10 mL of SC-Hismedia containing 2% galactose, and used to inoculate a total volume of100 mL of this media. Cultures were grown for 17 hours at 30° C. and 250rpm. Cells were then pelleted, rinsed in 0.85% NaCl, and repelleted.Cell pellets (190 mg) were suspended in 380 μL of 100 mM potassiumphosphate buffer, pH 7.5, and an equal volume (pellet plus buffer) ofpre-rinsed glass beads (Sigma, 150-212 microns) was added. This mixturewas vortexed for 30 seconds and placed on ice for 1 minute, and thevortexing/cooling cycle was repeated 7 additional times. The cells werethen centrifuged for 6 minutes at 5,000 g and the supernatant wasremoved to a fresh tube. The beads/pellet were washed twice with 300 μLof buffer and centrifuged, and the supernatants joined with the firstsupernatant.

An anaerobically-grown culture of E. coli strain DH10B was used as apositive control for D-LDH assays. A culture of this strain was grown tosaturation overnight and diluted 1:20 the following morning in fresh LBmedia. The culture was grown anaerobically at 37° C. for 7.5 hours.Cells were pelleted, washed with 0.85% NaCl, and repelleted. Cells weredisrupted using BugBuster™ Protein Extraction Reagent and Benzonase®(Novagen) as per manufacturer's instructions with a 20-minute incubationat room temperature. After centrifugation at 16,000 g and 4° C., thesupernatant was transferred to a new tube and used in the activityassay.

Total protein content of cell extracts was quantified using a microplateBio-Rad Protein Assay (Bio-Rad, Hercules, Calif.). The D-LDH andD-LDH/PCT constructs in YPH500, the pESC-His negative control in YPH500,and the anaerobically-grown E. coli strain were tested for their abilityto catalyze the conversion of pyruvate to lactate by assaying theconcurrent oxidation of NADH to NAD. The assay mixture contained 100 mMpotassium phosphate buffer, pH 7.5, 0.2 mM NADH, and 0.5-1.0 μg of cellextract. The reaction was started by the addition of sodium pyruvate toa final concentration of 5 mM, and the decrease in absorbance at 340 nmwas measured over 10 minutes. When 0.5 μg of total cell extract proteinof the negative control strain was added to the reaction mixture, adecrease in absorbance from −0.01 to −0.02 was seen over 200 seconds. Adecrease in absorbance from −0.21 to −0.47 and −0.20 to −0.47 over 200seconds was seen, respectively, for cell extract from theYPH500/pESC-His/D-LDH or YPH500/pESC-His/D-LDH/PCT strains. 0.5 μL (7.85μg) of cell extract from the anaerobically-grown E. coli strain showed adecrease in absorbance very similar to that for 1 μg of cell extract ofthe YPH500/pESC-His/D-LDH/PCT strain. When 4 μg of cell extract wasused, the YPH500/pESC-His/D-LDH/PCT strain showed a decrease inabsorbance from −0.18 to −0.60 over 10 minutes, whereas the negativecontrol strain showed no decrease in absorbance (−0.08 to −0.08).

D. Demonstration of 3-HP Production in S. cerevisiae

The pESC-Trp/OS19/EI, pESC-Leu/EIIa/EIIB, and pESC-His/D-LDH/PCTconstructs were transformed into a single strain of S. cerevisiae YPH500using a Frozen-EZ Yeast Transformation II™ Kit (Zymo Research, Orange,Calif.). A negative control strain was also developed by transformationof the pESC-Trp, pESC-Leu, and pESC-His vectors into a single YPH500strain. Transformation reactions were plated on SC-Trp-Leu-His media.Individual yeast colonies were screened by colony PCR for the presenceor absence of nucleic acid corresponding to each construct.

The strain carrying all six genes and the negative control strain weregrown in 5 mL of SC-Trp-Leu-His media containing 2% glucose. Thesecultures were grown for 31 hours at 30° C., and 2 mL was used toinoculate 50 mL of the same media. The subcultures were grown for 19hours at 30° C., and their OD600s were determined. For each strain, avolume of cells giving an OD×volume equal to 100 was pelleted, washedwith SC-Trp-Leu-His media with no carbon source, and repelleted. Thecells were suspended in 10 mL of SC-Trp-Leu-His media containing 2%galactose and 2% raffinose and used to inoculate a total volume of 250mL of this media. The cultures were grown in bottles at 30° C. with noshaking, and samples were taken at 0, 4.5, 20, 28.5, 45, and 70 hours.Samples were spun down to remove cells and the supernatant was filteredusing 0.45 micron Acrodisc Syrige Filters (Pall Gelman Laboratory, AnnArbor, Mich.).

100 microliters of the filtered broth was used to derive CoA esters ofany lactate or 3-HP in the broth using 6 micrograms of purifiedpropionyl-CoA transferase, 50 mM potassium phosphate buffer (pH 7.0),and 1 mM acetyl-CoA. The reaction was allowed to proceed at roomtemperature for 15 minutes and was stopped by adding 1 volume 10%trifluoroacetic acid. The reaction mixtures were purified using Sep PakC18 columns as previously described and analyzed by LC/MS.

Example 14 Constructing a Biosynthetic Pathway that Produces OrganicAcids from β-alanine

One possible pathway to 3-HP from β-alanine involves the use of apolypeptide having CoA transferase activity (e.g., an enzyme from aclass of enzymes that transfers a CoA group from one metabolite to theother). As shown in FIG. 54, β-alanine can be converted to β-alanyl-CoAusing a polypeptide having CoA transferase activity and CoA donors suchas acetyl-CoA or propionyl-CoA. Alternatively, α-alanyl-CoA can begenerated by the action of a polypeptide having CoA synthetase activity.The β-alanyl-CoA can be deaminated to form acrylyl-CoA by a polypeptidehaving α-alanyl-CoA ammonia lyase activity. The hydration of acrylyl-CoAat the β position to yield 3-HP-CoA can be carried out by a polypeptidehaving 3-HP-CoA dehydratase activity. The 3-HP-CoA can act as a CoAdonor for α-alanine, a reaction that can be catalyzed a polypeptidehaving CoA transferase activity, thus yielding 3-HP as a product.Alternatively, 3-HP-CoA can be hydrolyzed to yield 3-HP by a polypeptidehaving specific CoA hydrolase activity.

Methods for isolating, sequencing, expressing, and testing the activityof a polypeptide having CoA transferase activity are described herein.

A. Isolation of a Polypeptide Having D-alanyl-CoA Ammonia Lyase Activity

Polypeptides having β-alanyl-CoA ammonia lyase activity can catalyze theconversion of β-alanyl-CoA into acryly-CoA. The activity of suchpolypeptides has been described by Vagelos et al. (J. Biol. Chem.,234:490-497 (1959)) in Clostridum propionicum. This polypeptide can beused as part of the acrylate pathway in Clostridum propionicum toproduce propionic acid.

C. propionicum was grown at 37° C. in an anoxic medium containing 0.2%yeast extract, 0.2% trypticase peptone, 0.05% cysteine, 0.5% b-alanine,0.4% VRB-salts, 5 mM potassium phosphate, pH 7.0. The cells wereharvested after 12 hours and washed twice with 50 mM potassium phosphate(Kpi), pH 7.0. About 2 g of wet packed cells were re-suspended in 40 mLof Kpi, pH 7.0, 1 mM MgCl₂, 1 mM EDTA, and 1 mM DTT (Buffer A), andhomogenized by sonication at about 85-100 W power using a 3 mm tip(Branson sonifier 250). Cell debris was removed by centrifugation at100,000 g for 45 minutes in a Centricon T-1080 Ultra centrifuge, and thecell free extract (˜110 U/mg activity) was subjected to anion exchangechromatography on Source-15Q-material. The Source-15Q column was loadedwith 32 mL of cell free extract. The column was developed by a lineargradient of 0 M to 0.5 M NaCl within 10 column volumes. The polypeptideeluted between 70-110 mM NaCl.

The solution was adjusted to a final concentration of 1 M (NH₄)₂SO₄ andapplied onto a Resource-Phe column equilibrated with 1 M (NH₄)₂SO₄ inbuffer A. The polypeptide did not bind to this column.

The final preparation was obtained after concentration in an Amiconchamber (filter cut-off 30 kDa). The functional polypeptide is composedof four polypeptide sub-units, each having a molecular mass of 16 kDa.The polypeptide had a final specific activity of 1033 U/mg in thestandard assay (see below).

The polypeptide sample after every purification step was separated on a15% SDS-PAGE gel. The gel was stained with 0.1% Coomassie R 250, and thedestaining was achieved by using 7.1% acetic acid/5% ethanol solution.

The polypeptide was desalted by RP-HPLC and subjected to N-terminalsequencing by gas phase Edman degradation. The results of this analysisyielded a 35 amino acid N-terminal sequence of the polypeptide. Thesequence was as follows:

(SEQ ID NO:177) MV-GKKVVHHLMMSAKDAHYTGNLVNGARIVNQWGD.

B. Amplification of a Gene Fragment

The 35 amino acid sequence of the polypeptide having β-alanine-CoAammonia lyase activity was used to design primers with which to amplifythe corresponding DNA from genome of C. propionicium. Genomic DNA fromC. propionicum was isolated using the Gentra Genomic DNA isolation Kit(Gentra Systems, Minneapolis) following the genomic DNA protocol forgram-positive bacteria. A codon usage table for Clostridium propionicumwas used to back translate the seven amino acids on either end of theamino acid sequence to obtain 20-nucleotide degenerate primers:

ACLF: 5′-ATGGTWGGYAARAARGTWGT-3′ (SEQ ID NO:178) ACLR:5′-TCRCCCCAYTGRTTWACRAT-3′ (SEQ ID NO:179)

The primers were used in a 50 μL PCR reaction containing 1×Taq PCRbuffer, 0.6 μM each primer, 0.2 mM each dNTP, 2 units of Taq DNApolymerase (Roche Molecular Biochemicals, Indianapolis, Ind.), 2.5%(v/v) DMSO, and 100 ng of genomic DNA. PCR was conducted using atouchdown PCR program with 4 cycles at an annealing temperature of 58°C., 4 cycles at 56° C., 4 cycles at 54° C., and 24 cycles at 52° C. Eachcycle used an initial 30 second denaturing step at 94° C. and a 1.25minute extension at 72° C., and the program had an initial denaturationstep at 94° C. for 2 minutes and final extension at 72° C. for 5minutes. The amounts of PCR primer used in the reaction were increasedthree-fold above typical PCR amounts due to the amount of degeneracy inthe 3′ end of the primer. In addition, separate PCR reactions containingeach individual primer were made to identify PCR product resulting fromsingle degenerate primers. Twenty μL of each PCR product was separatedon a 2.0% TAE (Tris-acetate-EDTA)-agarose gel.

A band of about 100 bp was produced by the reaction containing both theforward and reverse primers, but was not present in the individualforward and reverse primer control reactions. This fragment was excisedand purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia,Calif.). Four microliters of the purified band was ligated intopCRII-TOPO vector and transformed by a heat-shock method into TOP10 E.coli cells using a TOPO cloning procedure (Invitrogen, Carlsbad,Calif.). Transformations were plated on LB media containing 50 μg/mL ofkanamycin and 50 μg/mL of5-Bromo-4-Chloro-3-Indolyl-B-D-Galactopyranoside (X-gal). Individual,white colonies were resuspended in 25 μL of 10 mM Tris and heated for 10minutes at 95° C. to break open the bacterial cells. Two microliters ofthe heated cells were used in a 25 μL PCR reaction using M13R and M13Funiversal primers homologous to the pCRII-TOPO vector. The PCR mixcontained the following: 1×Taq PCR buffer, 0.2 μM each primer, 0.2 mMeach dNTP, and 1 unit of Taq DNA polymerase per reaction. The PCRreaction was performed in a MJ Research PTC 100 under the followingconditions: an initial denaturation at 94° C. for 2 minutes; 30 cyclesof 94° C. for 30 seconds, 52° C. for 1 minute, and 72° C. for 1.25minutes; and a final extension for 7 minutes at 72° C. Plasmid DNA wasobtained (QIAprep Spin Miniprep Kit, Qiagen) from cultures of coloniesshowing the desired insert and was used for DNA sequencing with M13Runiversal primer. The following nucleic acid sequence was internal tothe degenerate primers and corresponds to a portion of the 35 amino acidresidue sequence:

(SEQ ID NO:180) 5′-ACATCATTTAATGATGA-GCGCAAAAGATGCTCACTATACTGGAAACTTAGTAAACGGCGCTAGA-3′.

C. Genome Walking to Obtain the Complete Coding Sequence

Primers for conducting genome walking in both upstream and downstreamdirections were designed using the portion of the nucleic acid sequencethat was internal to the degenerate primers. The primer sequences wereas follows:

(SEQ ID NO:181) ACLGSP1F: 5′-GTACATCATTTAATGATGAGCGCAAAAGATG-3′ (SEQ IDNO:182) ACLGSP2F: 5′-GATGCTCACTATACTGGAAACTTAGTAAAC-3′ (SEQ ID NO:183)ACLGSP1R: 5′-ATTCTAGCGCCGTTTACTAAGTTTCCAG-3′ (SEQ ID NO:184) ACLGSP2R:5′-CCAGTATAGTGAGCATCTTTTGCGCTCATC-3′

GSP1F and GSP2F are primers facing downstream, GSP1R and GSP2R areprimers facing upstream, and GSP2F and GSP2R are primers nested insideGSP1F and GSP1R, respectively. Genome walking libraries were constructedaccording to the manual for CLONTECH's Universal Genome Walking Kit(CLONTECH Laboratories, Palo Alto, Calif.), with the exception that therestriction enzymes Ssp I and Hinc II were used in addition to Dra I,EcoR V, and Pvu II. PCR was conducted in a Perkin Elmer 9700Thermocycler using the following reaction mix: 1×XL Buffer II, 0.2 mMeach dNTP, 1.25 mM Mg(OAc)₂, 0.2 μM each primer, 2 units of rTth DNApolymerase XL (Applied Biosystems, Foster City, Calif.), and 1 μL oflibrary per 50 μL reaction. First round PCR used an initial denaturationat 94° C. for 5 seconds; 7 cycles consisting of 2 sec at 94° C. and 3min at 70° C.; 32 cycles consisting of 2 sec at 94° C. and 3 min at 64°C.; and a final extension at 64° C. for 4 min. Second round PCR used aninitial denaturation at 94° C. for 15 seconds; 5 cycles consisting of 5sec at 94° C. and 3 min at 70° C.; 26 cycles consisting of 5 sec at 94°C. and 3 min at 64° C.; and a final extension at 66° C. for 7 min.Twenty μL of each first and second round product was run on a 1.0%TAE-agarose gel. In the second round PCR for the forward reactions, a1.4 Kb band was obtained for Dra I, a 1.5 Kb band for Hinc II, a 4.0 Kbband for Pvu II, and 2.0 and 2.6 Kb bands were obtained for Ssp I. Inthe second round PCR for the reverse reactions, a 1.5 Kb band wasobtained for Dra I, a 0.8 Kb band for EcoR V, a 2.0 Kb band for Hinc II,a 2.9 Kb band for Pvu II, and a 1.5 Kb band was obtained for Ssp I.Several of these fragments were gel purified, cloned, and sequenced.

The coding sequence of the polypeptide having β-alanyl-CoA ammonia lyaseactivity is set forth in SEQ ID NO:162. This coding sequence encodes theamino acid sequence set forth in SEQ ID NO:160. The coding sequence wascloned and expressed in bacterial cells. A polypeptide with the expectedsize was isolated and tested for enzymatic activity.

The isolation of a nucleic acid molecule encoding a polypeptide having3-HP-CoA dehydratase activity (e.g., the seventh enzymatic activity inFIG. 54, which can be accomplished with a polypeptide having the aminoacid sequence set forth in SEQ ID NO:41) is described herein. Thispolypeptide in combination with a polypeptide having CoA transferaseactivity (e.g., a polypeptide having the amino acid sequence set forthin SEQ ID NO:2) and a polypeptide having β-alanyl-CoA ammonia lyaseactivity (e.g., a polypeptide having the amino acid sequence set forthin SEQ ID NO: 160) provides one method of making 3-HP from β-alanine.

Example 15 Constructing a Biosynthetic Pathway that Produces OrganicAcids from β-alanine

In another pathway, β-alanine generated from aspartate can be deaminatedby a polypeptide having 4,4-aminobutyrate aminotransferase activity(FIG. 55). This reaction also can regenerate glutamate that is consumedin the formation of aspartate. The deamination of β-alanine can yieldmalonate semialdehyde, which can be further reduced to 3-HP by apolypeptide having 3-hydroxypropionate dehydrogenase activity or apolypeptide having 3-hydroxyisobutyrate dehydrogenase activity. Suchpolypeptides can be obtained as follows.

A. Cloning gabT (4-aminobutyrate aminotransferase) from C.acetobutycilicum

The following PCR primers were designed based on a published sequencefor a gabT gene from Clostridium acetobutycilicum (GenBank# AE007654):

Cac aba nco sen: 5′-GAGCCATGGAAGAAATAAATGCTAAAG-3′ (SEQ ID NO:185) Cacaba bam anti: 5′-AGAGGATGGCTTTTTAAATCGCTATTC-3′ (SEQ ID NO:186)

The primers introduced a NcoI site at the 5′ end and a BamH I site atthe 3′ end. A PCR reaction was set up using chromosomal DNA from C.acetobutylicum as the template.

H2O 80.75 μL PCR Program Taq Plus Long 10× Buffer 10 μL 94° C. 5 minutesdNTP mix (10 mM) 3 μL 25 cycles of: Cac aba nco sen (20 mM) 2 μL 94° C.30 seconds Cac aba bam anti (20 mM) 2 μL 50° C. 30 seconds C.acetobutylicum DNA 1 μL 72° C. 80 (~100 ng) seconds + 2 Taq Plus Long (5U/mL) 1 μL seconds/cycle Pfu (2.5 U/mL) 0.25 μL 1 cycle of: 68° C. 7minutes  4° C. until use

Upon agarose gel analysis a single band was observed of ˜1.3 Kb in size.This fragment was purified using QIAquick PCR purification kit (Qiagen,Valencia, Calif.) and cloned into pCRII TOPO using the TOPO Zero BluntPCR cloning kit (Invitrogen, Carlsbad, Calif.). 1 μL of the pCRII TOPOligation mix was used to transform chemically competent TOP10 E. colicells. The cells were for 1 hour in SOC media, and the transformantswere selected on LB/kanamycin (50 μg/mL) plates. Single colonies of thetransformant grown overnight in LB/kanamycin media, and the plasmid DNAwas extracted using a Mini prep kit (Qiagen, Valencia, Calif.). Thesuper-coiled plasmid DNA was separated on a 1% agarose gel digested, andthe colonies with insert were selected. The insert was sequenced toconfirm the sequence and its quality.

The plasmid having the correct insert was digested with restrictionenzyme Nco I and BamH I. The digested insert was gel isolated andligated to pET28b expression vector that was also restricted with Nco Iand BamH I enzymes. 1 μl of ligation mix was used to transformchemically competent TOP10 E. coli cells. The cells were recovered for 1hour in SOC media, and the transformants were selected on LB/kanamycin(50 μg/mL) plates. The super-coiled plasmid DNA was separated on a 1%agarose gel digested, and the colonies with insert were selected. Theplasmid with the insert was isolated using a Mini prep kit (Qiagen,Valencia, Calif.), and 1 μL of this plasmid DNA was used to transformelectrocompetent BL21(DE3) (Novagen, Madison, Wis.). These cells wereused to check the expression of a polypeptide having 4-aminobutyrateaminotransferase activity.

B. Cloning mmsB (3-hydroxyisobutyrate dehydrogenase) from P. aeruginosa

The following PCR primers was designed based on a published sequence fora mmsB gene from Pseudomona aeruginosa (GenBank# M84911):

Ppu hid nde sen: 5′-ATACATATGACCGACCGACATCGCATT-3′ (SEQ ID NO:186) Ppuhid sal anti: 5′-ATAGTCGACGGGTCAGTCCTTGCCGCG-3′ (SEQ ID NO:187)

The primers introduced a Nde I site at the 5′ end and a BamH I site atthe 3′ end.

H₂O 80.75 μL    PCR Program Taq Plus Long 10× Buffer 10 μL  94° C. 5minutes dNTP mix (10 mM) 3 μL 25 cycles of: 94° C. 30 seconds 55° C. 30seconds 72° C. 90 seconds + 2 seconds/cycle Ppu hid nde sen (20 μM) 2 μL68° C. 7 minutes Ppu hid sal anti (20 μM) 2 μL  4° C. until use C.acetobutylicum DNA 1 μl (~100 ng) Taq Plus Long (Stratagene, 1 μL LaJolla, CA) Pfu (Stratagene, La Jolla, CA) 0.25 μL  

A PCR reaction was set up using chromosomal DNA from P. aeruginosa asthe template. Chromosomal DNA was obtained from ATCC (Manassas, Va.) P.aeruginosa 17933D.

Upon agarose gel analysis, a single band was observed of ˜1.6 Kb insize. This fragment was purified using QIAquick PCR purification kit(Qiagen, Valencia, Calif.) and cloned into pCRII TOPO using the TOPOZero Blunt PCR cloning kit (Invitrogen, Carlsbad, Calif.). 1 μL of thepCRII TOPO ligation mix was used to transform chemically competent TOP10E. coli cells. The cells were recovered for 1 hour in SOC media, and thetransformants were selected on LB/kanamycin (50 μg/mL) plates. Singlecolonies of the transformant grown overnight in LB/kanamycin media, andthe plasmid DNA was extracted using a Mini prep kit (Qiagen, Valencia,Calif.). The super-coiled plasmid DNA was separated on a 1% agarose geland digested, and the colonies with insert were selected. The insert wassequenced to confirm the sequence and its quality.

The plasmid having the correct insert was digested with restrictionenzyme Nde I and BamHI. The digested insert was gel isolated and ligatedto pET30a expression vector that was also restricted with Nde I and BamHI enzymes. 1 μL of ligation mix was used to transform chemicallycompetent TOP10 E. coli cells. The cells were recovered for 1 hour inSOC media, and the transformants were selected on LB/kanamycin (50μg/mL) plates. The super-coiled plasmid DNA was separated on a 1%agarose gel and digested, and the colonies with insert were selected.The plasmid with the insert was isolated using a Mini prep kit (Qiagen,Valencia, Calif.), and 1 μl of this plasmid DNA was used to transformelectrocompetent BL21(DE3) (Novagen, Madison, Wis.). These cells wereused to check the expression of a polypeptide having3-hydroxyisobutyrate dehydrogenase activity.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. An isolated cell comprising: lactyl-CoA dehydratase activity;3-hydroxypropionyl-CoA dehydratase activity; and poly hydroxyacidsynthase activity.
 2. The isolated cell of claim 1, wherein said cellproduces polymerized 3-HP.
 3. The isolated cell of claim 1, wherein saidcell is prokaryotic.
 4. The isolated cell of claim 1, wherein said cellis selected from the group consisting of yeast, Lactobacillus,Lactococcus, Bacillus, and Escherichia cells.
 5. The isolated cell ofclaim 1, wherein said poly hydroxyacid synthase activity is encoded by anucleic acid sequence comprising a sequence as set forth in GenBankaccession number X97200.
 6. The isolated cell of claim 1, furthercomprising CoA synthetase activity.
 7. A transformed cell comprising atleast one exogenous nucleic acid molecule encoding CoA synthetase, CoAtransferase, lactyl-CoA dehydratase, 3-hydroxypropionyl-CoA dehydrataseand/or poly hydroxyacid synthase.
 8. The transformed cell of claim 7,wherein the cell produces polymerized 3-HP.
 9. The transformed cell ofclaim 7, wherein said cell is prokaryotic.
 10. The transformed cell ofclaim 7, wherein said cell is selected from the group consisting ofLactobacillus, Lactococcus, Bacillus, and Escherichia cells.
 11. Thetransformed cell of claim 7, wherein the cell is a yeast cell.
 12. Amethod for making polymerized 3-HP, comprising: culturing a cell underconditions wherein said cell produces said polymerized 3-HP, said cellcomprising lactyl-CoA dehydratase activity and 3-hydroxypropionyl-CoAdehydratase activity.
 13. The method of claim 12, wherein said cell isselected from the group consisting of yeast, Lactobacillus, Lactococcus,Bacillus, and Escherichia cells.
 14. The method of claim 12, whereinsaid cell further comprises CoA synthetase activity.
 15. The method ofclaim 12, wherein said cell further comprises poly hydroxyacid synthaseactivity.
 16. A method for making polymerized 3-HP, comprising: a)contacting lactate with a first polypeptide having CoA synthetase or CoAtransferase activity to form lactyl-CoA, b) contacting said lactyl-CoAwith a second polypeptide having lactyl-CoA dehydratase activity to formacrylyl-CoA, c) contacting said acrylyl-CoA with a third polypeptidehaving 3-hydroxypropionyl-CoA dehydratase activity to form3-hydroxypropionic acid-CoA, and d) contacting said 3-hydroxypropionicacid-CoA with a fourth polypeptide having poly hydroxyacid synthaseactivity to form said polymerized 3-HP.
 17. A method for makingpolymerized 3-HP, comprising: culturing a cell under conditions whereinsaid cell produces said polymerized 3-HP, said cell comprising at leastone exogenous nucleic acid that encodes at least one polypeptide suchthat said polymerized 3-HP is produced from 3-HP-CoA and underconditions such that said polymerized 3-HP is produced.
 18. The methodof claim 17, wherein said cell is selected from the group consisting ofyeast, Lactobacillus, Lactococcus, Bacillus, and Escherichia cells.